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Amyloid beta dimers/trimers potently induce cofilin-actin rods that are inhibited by maintaining cofilin-phosphorylation.

Davis RC, Marsden IT, Maloney MT, Minamide LS, Podlisny M, Selkoe DJ, Bamburg JR - Mol Neurodegener (2011)

Bottom Line: Fractions from 7PA2 medium containing Aβ monomers are not active, suggesting oxidized SDS-stable Aβ1-42 dimers in a low-n state are the most active rod-inducing species.Overexpression of cofilin phosphatases increase rod formation when expressed alone and exacerbate rod formation when coupled with Aβd/t, whereas overexpression of a cofilin kinase inhibits Aβd/t-induced rod formation.Together these data support a mechanism by which Aβd/t alters the actin cytoskeleton via effects on cofilin in neurons critical to learning and memory.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523-1870, USA. jbamburg@lamar.colostate.edu.

ABSTRACT

Background: Previously we reported 1 μM synthetic human amyloid beta1-42 oligomers induced cofilin dephosphorylation (activation) and formation of cofilin-actin rods within rat hippocampal neurons primarily localized to the dentate gyrus.

Results: Here we demonstrate that a gel filtration fraction of 7PA2 cell-secreted SDS-stable human Aβ dimers and trimers (Aβd/t) induces maximal neuronal rod response at ~250 pM. This is 4,000-fold more active than traditionally prepared human Aβ oligomers, which contain SDS-stable trimers and tetramers, but are devoid of dimers. When incubated under tyrosine oxidizing conditions, synthetic human but not rodent Aβ1-42, the latter lacking tyrosine, acquires a marked increase (620 fold for EC50) in rod-inducing activity. Gel filtration of this preparation yielded two fractions containing SDS-stable dimers, trimers and tetramers. One, eluting at a similar volume to 7PA2 Aβd/t, had maximum activity at ~5 nM, whereas the other, eluting at the void volume (high-n state), lacked rod inducing activity at the same concentration. Fractions from 7PA2 medium containing Aβ monomers are not active, suggesting oxidized SDS-stable Aβ1-42 dimers in a low-n state are the most active rod-inducing species. Aβd/t-induced rods are predominantly localized to the dentate gyrus and mossy fiber tract, reach significance over controls within 2 h of treatment, and are reversible, disappearing by 24 h after Aβd/t washout. Overexpression of cofilin phosphatases increase rod formation when expressed alone and exacerbate rod formation when coupled with Aβd/t, whereas overexpression of a cofilin kinase inhibits Aβd/t-induced rod formation.

Conclusions: Together these data support a mechanism by which Aβd/t alters the actin cytoskeleton via effects on cofilin in neurons critical to learning and memory.

No MeSH data available.


Related in: MedlinePlus

Oxidized cross-linking of synthetic Aβ1-42 generates a dimer that potently induces rods. (A) Western blot showing SDS-stable species of Aβ in different preparations. Two gel filtration fractions from medium of 7PA2 cells are combined to give Aβd/t. Last three lanes are synthetic human Aβ1-42. Lane 1: traditional oligomers prepared in DMSO/F12 [44,45]; Lane 2: peptide incubated (37°C, 3 d) in PBS containing 250 μM H2O2; Lane 3: peptide incubated (5 d) in PBS containing 250 μM peroxide plus 25 μM CuCl2. Dimer is absent in traditional oligomer preparations but forms in peroxide alone. Dimer, trimer and tetramer are generated with Cu2+/peroxide. (B) Comparison of rod-inducing activity between untreated (Untr) dissociated neurons and neurons treated with: Cu2+/peroxide (veh cont), 1 μM traditional synthetic human Aβ oligomers (HAβsyn), 1 μM synthetic rodent Aβ (RAβsyn) treated identically to HAβsyn, 1 μM Cu2+/peroxide-treated RAβsyn, 10 nM (45 ng/mL) and 1 nM peroxide-oxidized HAβsyn, and 5 nM each of high-n and low-n oligomers-containing fractions of oxidized HAβsyn (see Additional file 3). (C) The rod-inducing activity of different Aβ preparations. The effective concentration for a 50% maximal response (EC50; arrows) was calculated from dose-response curves for traditional Aβ oligomers (Aβsyn; ED50 = 3100 ng/mL), Cu2+/peroxide oxidized synthetic Aβ (OxAβsyn; ED50 = 5 ng/mL) and Aβd/t (ED50 = 0.4 ng/mL). Dashed line is control (untreated). Bars are standard deviations. (D) Human and rodent Aβ1-42 sequences differ in three residues (bold), but only tyrosine at position 10 is likely to generate the peroxide-induced SDS-stable species.
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Figure 3: Oxidized cross-linking of synthetic Aβ1-42 generates a dimer that potently induces rods. (A) Western blot showing SDS-stable species of Aβ in different preparations. Two gel filtration fractions from medium of 7PA2 cells are combined to give Aβd/t. Last three lanes are synthetic human Aβ1-42. Lane 1: traditional oligomers prepared in DMSO/F12 [44,45]; Lane 2: peptide incubated (37°C, 3 d) in PBS containing 250 μM H2O2; Lane 3: peptide incubated (5 d) in PBS containing 250 μM peroxide plus 25 μM CuCl2. Dimer is absent in traditional oligomer preparations but forms in peroxide alone. Dimer, trimer and tetramer are generated with Cu2+/peroxide. (B) Comparison of rod-inducing activity between untreated (Untr) dissociated neurons and neurons treated with: Cu2+/peroxide (veh cont), 1 μM traditional synthetic human Aβ oligomers (HAβsyn), 1 μM synthetic rodent Aβ (RAβsyn) treated identically to HAβsyn, 1 μM Cu2+/peroxide-treated RAβsyn, 10 nM (45 ng/mL) and 1 nM peroxide-oxidized HAβsyn, and 5 nM each of high-n and low-n oligomers-containing fractions of oxidized HAβsyn (see Additional file 3). (C) The rod-inducing activity of different Aβ preparations. The effective concentration for a 50% maximal response (EC50; arrows) was calculated from dose-response curves for traditional Aβ oligomers (Aβsyn; ED50 = 3100 ng/mL), Cu2+/peroxide oxidized synthetic Aβ (OxAβsyn; ED50 = 5 ng/mL) and Aβd/t (ED50 = 0.4 ng/mL). Dashed line is control (untreated). Bars are standard deviations. (D) Human and rodent Aβ1-42 sequences differ in three residues (bold), but only tyrosine at position 10 is likely to generate the peroxide-induced SDS-stable species.

Mentions: Oligomerization of human Aβsyn is usually performed on peptide solubilized in DMSO [44,45]. These oligomer preparations are noticeably deficient in SDS-stable dimer [59], although SDS-stable trimers and tetramers are present (Figure 3A). Therefore we reasoned that it is likely the increased dimer content that makes the Aβd/t so much more active in rod-induction than Aβsyn. Oxidation methods to prepare stable tyrosine cross-linked human Aβ dimer have been reported [46]. Human Aβ has a single tyrosine at residue 10, which is substituted by phenylalanine in rodent Aβ (Figure 3D). Rodent Aβ (RAβ) does not form dimers or other SDS-stable oligomers on its own [46] and does not induce rods even when used at 1 μM concentration (Figure 3B). Thus, we addressed the possibility that oxidized human Aβ dimers are contributing to the enhanced rod forming activity of the Aβd/t fraction. Synthetic human Aβ1-42 was incubated with 250 μM hydrogen peroxide plus or minus 25 μM CuCl2 for 3-5 days (Additional file 2). The presence of the CuCl2 is required to generate the di-tyrosine dimer [46] but not other oxidation products that include dimers (Figure 3A). We compared the rod-inducing ability of the Cu2+/peroxide oxidized synthetic human Aβ (OxAβsyn) to that of the Aβd/t and the traditionally prepared synthetic human Aβ oligomers (Aβsyn) by performing dose-response curves in cultures of rat hippocampal neurons treated on day 4 and fixed on day 5 (Figure 3C). The presence of Cu2+/peroxide (Figure 3B) or peroxide alone (data not shown) had no effect on rod formation. It is quite apparent from the curves in Figure 3C that Aβ oxidation dramatically enhances its rod-inducing activity. The effective concentration for half-maximal response (EC50) is >7000 fold different between Aβsyn (3100 ng/mL = 690 nM) and Aβd/t (0.4 ng/mL = 90 pM) and >600 fold different between Aβsyn and OxAβsyn (5 ng/mL = 1.1 nM). These concentrations are based on the total Aβ content in the samples tested. If we subtract the amount of monomer remaining in each of the fractions based upon its percentage content from quantified Western blots, there is less than a five-fold difference in the activity of the OxAβsyn and Aβd/t samples. In addition, synthetic rodent Aβ1-42 (RAβsyn) is inactive in rod induction at 1 μM when prepared using either standard oligomerizing conditions (DMSO/F12 medium) or Cu2+/peroxide treatment (Figure 3B), strongly suggesting that oxidation of tyrosine is what drives much, if not all, of the formation of SDS-stable oligomers. Gel filtration of the Cu2+/peroxide-oxidized human Aβsyn produced two peaks containing Aβ (Additional file 3). Western blots showed that both peaks contained similar mixtures of SDS-stable species (monomer, dimer, trimer and tetramer) with about 25% eluting near the void volume (high-n species) and the remaining 75% eluting near the position of 7PA2 cell-secreted Aβd/t (low-n species), which elutes at the position of dimer, based upon globular protein calibration (Additional file 3). The high-n fractions were inactive for rod-inducing activity at 23 ng/mL (5 nM), whereas the low-n fractions at the same concentration gave as strong a response as has been obtained with any Aβ species (Figure 3B). Taken together, these results suggest that a variety of oxidized human Aβ dimers can form, that their formation appears to be dependent upon the tyrosine at position 10, and that their rod-inducing activity may be dramatically impacted by their final aggregation state. The presence of the SDS-stable Aβ dimer in a low-n state appears to be largely responsible for the rod-inducing potency of the Aβ.


Amyloid beta dimers/trimers potently induce cofilin-actin rods that are inhibited by maintaining cofilin-phosphorylation.

Davis RC, Marsden IT, Maloney MT, Minamide LS, Podlisny M, Selkoe DJ, Bamburg JR - Mol Neurodegener (2011)

Oxidized cross-linking of synthetic Aβ1-42 generates a dimer that potently induces rods. (A) Western blot showing SDS-stable species of Aβ in different preparations. Two gel filtration fractions from medium of 7PA2 cells are combined to give Aβd/t. Last three lanes are synthetic human Aβ1-42. Lane 1: traditional oligomers prepared in DMSO/F12 [44,45]; Lane 2: peptide incubated (37°C, 3 d) in PBS containing 250 μM H2O2; Lane 3: peptide incubated (5 d) in PBS containing 250 μM peroxide plus 25 μM CuCl2. Dimer is absent in traditional oligomer preparations but forms in peroxide alone. Dimer, trimer and tetramer are generated with Cu2+/peroxide. (B) Comparison of rod-inducing activity between untreated (Untr) dissociated neurons and neurons treated with: Cu2+/peroxide (veh cont), 1 μM traditional synthetic human Aβ oligomers (HAβsyn), 1 μM synthetic rodent Aβ (RAβsyn) treated identically to HAβsyn, 1 μM Cu2+/peroxide-treated RAβsyn, 10 nM (45 ng/mL) and 1 nM peroxide-oxidized HAβsyn, and 5 nM each of high-n and low-n oligomers-containing fractions of oxidized HAβsyn (see Additional file 3). (C) The rod-inducing activity of different Aβ preparations. The effective concentration for a 50% maximal response (EC50; arrows) was calculated from dose-response curves for traditional Aβ oligomers (Aβsyn; ED50 = 3100 ng/mL), Cu2+/peroxide oxidized synthetic Aβ (OxAβsyn; ED50 = 5 ng/mL) and Aβd/t (ED50 = 0.4 ng/mL). Dashed line is control (untreated). Bars are standard deviations. (D) Human and rodent Aβ1-42 sequences differ in three residues (bold), but only tyrosine at position 10 is likely to generate the peroxide-induced SDS-stable species.
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Figure 3: Oxidized cross-linking of synthetic Aβ1-42 generates a dimer that potently induces rods. (A) Western blot showing SDS-stable species of Aβ in different preparations. Two gel filtration fractions from medium of 7PA2 cells are combined to give Aβd/t. Last three lanes are synthetic human Aβ1-42. Lane 1: traditional oligomers prepared in DMSO/F12 [44,45]; Lane 2: peptide incubated (37°C, 3 d) in PBS containing 250 μM H2O2; Lane 3: peptide incubated (5 d) in PBS containing 250 μM peroxide plus 25 μM CuCl2. Dimer is absent in traditional oligomer preparations but forms in peroxide alone. Dimer, trimer and tetramer are generated with Cu2+/peroxide. (B) Comparison of rod-inducing activity between untreated (Untr) dissociated neurons and neurons treated with: Cu2+/peroxide (veh cont), 1 μM traditional synthetic human Aβ oligomers (HAβsyn), 1 μM synthetic rodent Aβ (RAβsyn) treated identically to HAβsyn, 1 μM Cu2+/peroxide-treated RAβsyn, 10 nM (45 ng/mL) and 1 nM peroxide-oxidized HAβsyn, and 5 nM each of high-n and low-n oligomers-containing fractions of oxidized HAβsyn (see Additional file 3). (C) The rod-inducing activity of different Aβ preparations. The effective concentration for a 50% maximal response (EC50; arrows) was calculated from dose-response curves for traditional Aβ oligomers (Aβsyn; ED50 = 3100 ng/mL), Cu2+/peroxide oxidized synthetic Aβ (OxAβsyn; ED50 = 5 ng/mL) and Aβd/t (ED50 = 0.4 ng/mL). Dashed line is control (untreated). Bars are standard deviations. (D) Human and rodent Aβ1-42 sequences differ in three residues (bold), but only tyrosine at position 10 is likely to generate the peroxide-induced SDS-stable species.
Mentions: Oligomerization of human Aβsyn is usually performed on peptide solubilized in DMSO [44,45]. These oligomer preparations are noticeably deficient in SDS-stable dimer [59], although SDS-stable trimers and tetramers are present (Figure 3A). Therefore we reasoned that it is likely the increased dimer content that makes the Aβd/t so much more active in rod-induction than Aβsyn. Oxidation methods to prepare stable tyrosine cross-linked human Aβ dimer have been reported [46]. Human Aβ has a single tyrosine at residue 10, which is substituted by phenylalanine in rodent Aβ (Figure 3D). Rodent Aβ (RAβ) does not form dimers or other SDS-stable oligomers on its own [46] and does not induce rods even when used at 1 μM concentration (Figure 3B). Thus, we addressed the possibility that oxidized human Aβ dimers are contributing to the enhanced rod forming activity of the Aβd/t fraction. Synthetic human Aβ1-42 was incubated with 250 μM hydrogen peroxide plus or minus 25 μM CuCl2 for 3-5 days (Additional file 2). The presence of the CuCl2 is required to generate the di-tyrosine dimer [46] but not other oxidation products that include dimers (Figure 3A). We compared the rod-inducing ability of the Cu2+/peroxide oxidized synthetic human Aβ (OxAβsyn) to that of the Aβd/t and the traditionally prepared synthetic human Aβ oligomers (Aβsyn) by performing dose-response curves in cultures of rat hippocampal neurons treated on day 4 and fixed on day 5 (Figure 3C). The presence of Cu2+/peroxide (Figure 3B) or peroxide alone (data not shown) had no effect on rod formation. It is quite apparent from the curves in Figure 3C that Aβ oxidation dramatically enhances its rod-inducing activity. The effective concentration for half-maximal response (EC50) is >7000 fold different between Aβsyn (3100 ng/mL = 690 nM) and Aβd/t (0.4 ng/mL = 90 pM) and >600 fold different between Aβsyn and OxAβsyn (5 ng/mL = 1.1 nM). These concentrations are based on the total Aβ content in the samples tested. If we subtract the amount of monomer remaining in each of the fractions based upon its percentage content from quantified Western blots, there is less than a five-fold difference in the activity of the OxAβsyn and Aβd/t samples. In addition, synthetic rodent Aβ1-42 (RAβsyn) is inactive in rod induction at 1 μM when prepared using either standard oligomerizing conditions (DMSO/F12 medium) or Cu2+/peroxide treatment (Figure 3B), strongly suggesting that oxidation of tyrosine is what drives much, if not all, of the formation of SDS-stable oligomers. Gel filtration of the Cu2+/peroxide-oxidized human Aβsyn produced two peaks containing Aβ (Additional file 3). Western blots showed that both peaks contained similar mixtures of SDS-stable species (monomer, dimer, trimer and tetramer) with about 25% eluting near the void volume (high-n species) and the remaining 75% eluting near the position of 7PA2 cell-secreted Aβd/t (low-n species), which elutes at the position of dimer, based upon globular protein calibration (Additional file 3). The high-n fractions were inactive for rod-inducing activity at 23 ng/mL (5 nM), whereas the low-n fractions at the same concentration gave as strong a response as has been obtained with any Aβ species (Figure 3B). Taken together, these results suggest that a variety of oxidized human Aβ dimers can form, that their formation appears to be dependent upon the tyrosine at position 10, and that their rod-inducing activity may be dramatically impacted by their final aggregation state. The presence of the SDS-stable Aβ dimer in a low-n state appears to be largely responsible for the rod-inducing potency of the Aβ.

Bottom Line: Fractions from 7PA2 medium containing Aβ monomers are not active, suggesting oxidized SDS-stable Aβ1-42 dimers in a low-n state are the most active rod-inducing species.Overexpression of cofilin phosphatases increase rod formation when expressed alone and exacerbate rod formation when coupled with Aβd/t, whereas overexpression of a cofilin kinase inhibits Aβd/t-induced rod formation.Together these data support a mechanism by which Aβd/t alters the actin cytoskeleton via effects on cofilin in neurons critical to learning and memory.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO 80523-1870, USA. jbamburg@lamar.colostate.edu.

ABSTRACT

Background: Previously we reported 1 μM synthetic human amyloid beta1-42 oligomers induced cofilin dephosphorylation (activation) and formation of cofilin-actin rods within rat hippocampal neurons primarily localized to the dentate gyrus.

Results: Here we demonstrate that a gel filtration fraction of 7PA2 cell-secreted SDS-stable human Aβ dimers and trimers (Aβd/t) induces maximal neuronal rod response at ~250 pM. This is 4,000-fold more active than traditionally prepared human Aβ oligomers, which contain SDS-stable trimers and tetramers, but are devoid of dimers. When incubated under tyrosine oxidizing conditions, synthetic human but not rodent Aβ1-42, the latter lacking tyrosine, acquires a marked increase (620 fold for EC50) in rod-inducing activity. Gel filtration of this preparation yielded two fractions containing SDS-stable dimers, trimers and tetramers. One, eluting at a similar volume to 7PA2 Aβd/t, had maximum activity at ~5 nM, whereas the other, eluting at the void volume (high-n state), lacked rod inducing activity at the same concentration. Fractions from 7PA2 medium containing Aβ monomers are not active, suggesting oxidized SDS-stable Aβ1-42 dimers in a low-n state are the most active rod-inducing species. Aβd/t-induced rods are predominantly localized to the dentate gyrus and mossy fiber tract, reach significance over controls within 2 h of treatment, and are reversible, disappearing by 24 h after Aβd/t washout. Overexpression of cofilin phosphatases increase rod formation when expressed alone and exacerbate rod formation when coupled with Aβd/t, whereas overexpression of a cofilin kinase inhibits Aβd/t-induced rod formation.

Conclusions: Together these data support a mechanism by which Aβd/t alters the actin cytoskeleton via effects on cofilin in neurons critical to learning and memory.

No MeSH data available.


Related in: MedlinePlus