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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

Percent of neurons in dissociated hippocampal cultures containing rods as a function of Aβ form, concentration and time of treatment. (A) Dose-response curves for Aβsyn oligomers and Aβd/t versus control. The concentrations are expressed in terms of the 7PA2 CHO cell secreted concentration of Aβd/t (1X = 250 pM), which was used at 0.1, 0.5 and 2X this value. For the Aβsyn oligomers, the 1X value equals 1.0 μM. (B) Following treatment with 1X amounts of Aβd/t or Aβsyn oligomers, neurons were fixed at the times shown and the percent of neurons with rods was quantified. By 2 h the percent of neurons forming rods in response to Aβd/t was significant (*) over controls (p = 0.05). The Aβsyn-treated neurons required 6 h to reach significance over controls. Significance (# = p = 0.05) in the differences between the two Aβ species occurred at 8 h.
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Figure 2: Percent of neurons in dissociated hippocampal cultures containing rods as a function of Aβ form, concentration and time of treatment. (A) Dose-response curves for Aβsyn oligomers and Aβd/t versus control. The concentrations are expressed in terms of the 7PA2 CHO cell secreted concentration of Aβd/t (1X = 250 pM), which was used at 0.1, 0.5 and 2X this value. For the Aβsyn oligomers, the 1X value equals 1.0 μM. (B) Following treatment with 1X amounts of Aβd/t or Aβsyn oligomers, neurons were fixed at the times shown and the percent of neurons with rods was quantified. By 2 h the percent of neurons forming rods in response to Aβd/t was significant (*) over controls (p = 0.05). The Aβsyn-treated neurons required 6 h to reach significance over controls. Significance (# = p = 0.05) in the differences between the two Aβ species occurred at 8 h.

Mentions: We next compared Aβd/t to Aβsyn oligomers for dose-response in rod-induction. The gel filtration fractions containing Aβd/t elute in 50 mM ammonium acetate, pH 8.5 and are freeze dried to remove most of the volatile buffer. However, when reconstituted they cannot be used above a 2.5X concentration because of increased cell death (release of LDH, data not shown). However, neurons could be treated with the Aβd/t from 0.1X to 2X (25 pM to 500 pM) without any significant cell loss over 48 h. Controls were untreated neurons or neurons treated with NC medium. As shown in Figure 2A, the half-maximal response in terms of numbers of neurons forming rods is achieved with ~0.4X Aβd/t (~100 pM) and ~0.7 μM Aβsyn oligomers, about a 7000 fold difference. This value compares favorably with the 4000 fold difference obtained using single point comparison at 1X concentration (250 pM Aβd/t versus 1 μM Aβsyn oligomers), which gives near maximal rod response for each preparation. Furthermore, the maximum percentage of cells with rods is greater with Aβd/t than with Aβsyn oligomers at all concentrations tested (Figure 2A).


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)

Percent of neurons in dissociated hippocampal cultures containing rods as a function of Aβ form, concentration and time of treatment. (A) Dose-response curves for Aβsyn oligomers and Aβd/t versus control. The concentrations are expressed in terms of the 7PA2 CHO cell secreted concentration of Aβd/t (1X = 250 pM), which was used at 0.1, 0.5 and 2X this value. For the Aβsyn oligomers, the 1X value equals 1.0 μM. (B) Following treatment with 1X amounts of Aβd/t or Aβsyn oligomers, neurons were fixed at the times shown and the percent of neurons with rods was quantified. By 2 h the percent of neurons forming rods in response to Aβd/t was significant (*) over controls (p = 0.05). The Aβsyn-treated neurons required 6 h to reach significance over controls. Significance (# = p = 0.05) in the differences between the two Aβ species occurred at 8 h.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3037337&req=5

Figure 2: Percent of neurons in dissociated hippocampal cultures containing rods as a function of Aβ form, concentration and time of treatment. (A) Dose-response curves for Aβsyn oligomers and Aβd/t versus control. The concentrations are expressed in terms of the 7PA2 CHO cell secreted concentration of Aβd/t (1X = 250 pM), which was used at 0.1, 0.5 and 2X this value. For the Aβsyn oligomers, the 1X value equals 1.0 μM. (B) Following treatment with 1X amounts of Aβd/t or Aβsyn oligomers, neurons were fixed at the times shown and the percent of neurons with rods was quantified. By 2 h the percent of neurons forming rods in response to Aβd/t was significant (*) over controls (p = 0.05). The Aβsyn-treated neurons required 6 h to reach significance over controls. Significance (# = p = 0.05) in the differences between the two Aβ species occurred at 8 h.
Mentions: We next compared Aβd/t to Aβsyn oligomers for dose-response in rod-induction. The gel filtration fractions containing Aβd/t elute in 50 mM ammonium acetate, pH 8.5 and are freeze dried to remove most of the volatile buffer. However, when reconstituted they cannot be used above a 2.5X concentration because of increased cell death (release of LDH, data not shown). However, neurons could be treated with the Aβd/t from 0.1X to 2X (25 pM to 500 pM) without any significant cell loss over 48 h. Controls were untreated neurons or neurons treated with NC medium. As shown in Figure 2A, the half-maximal response in terms of numbers of neurons forming rods is achieved with ~0.4X Aβd/t (~100 pM) and ~0.7 μM Aβsyn oligomers, about a 7000 fold difference. This value compares favorably with the 4000 fold difference obtained using single point comparison at 1X concentration (250 pM Aβd/t versus 1 μM Aβsyn oligomers), which gives near maximal rod response for each preparation. Furthermore, the maximum percentage of cells with rods is greater with Aβd/t than with Aβsyn oligomers at all concentrations tested (Figure 2A).

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