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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

Numbers of rods induced by Aβd/t are highest in the dentate gyrus and mossy fiber tract. (A) Organotypic hippocampal slices were cultured for at least 8-10 days and were left untreated or treated with 1X Aβd/t, the same amount of the equivalent NC medium fraction, or scrambled Aβ peptide. After 48 h, slices were fixed and immunostained for cofilin and DNA (DAPI), and rods were quantified by counting with a 60x objective. Rod mapping from multiple slices onto a matrix grid of the hippocampus was performed as previously described using fiduciary markers from the stained nuclei layers to align hippocampal regions [29]. There were no differences detected in rod numbers or distribution between the untreated slices and those treated with NC medium or scrambled peptide and these were all combined to give the control panel. Rods induced by the Aβd/t were heavily concentrated over the dentate gyrus and mossy fiber tract (n = 18 for control slices and n = 12 for Aβd/t treated). (B) Rod quantification averaged per field over different regions of the slices. Each field acquired with the 60x objective has about 6-7 matrix grid squares. The only regions of significance (# = p = 0.05) for the rod numbers are in the dentate gyrus and mossy fiber tract.
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Figure 4: Numbers of rods induced by Aβd/t are highest in the dentate gyrus and mossy fiber tract. (A) Organotypic hippocampal slices were cultured for at least 8-10 days and were left untreated or treated with 1X Aβd/t, the same amount of the equivalent NC medium fraction, or scrambled Aβ peptide. After 48 h, slices were fixed and immunostained for cofilin and DNA (DAPI), and rods were quantified by counting with a 60x objective. Rod mapping from multiple slices onto a matrix grid of the hippocampus was performed as previously described using fiduciary markers from the stained nuclei layers to align hippocampal regions [29]. There were no differences detected in rod numbers or distribution between the untreated slices and those treated with NC medium or scrambled peptide and these were all combined to give the control panel. Rods induced by the Aβd/t were heavily concentrated over the dentate gyrus and mossy fiber tract (n = 18 for control slices and n = 12 for Aβd/t treated). (B) Rod quantification averaged per field over different regions of the slices. Each field acquired with the 60x objective has about 6-7 matrix grid squares. The only regions of significance (# = p = 0.05) for the rod numbers are in the dentate gyrus and mossy fiber tract.

Mentions: The numbers of rods in fields taken from multiple organotypic hippocampal slices that were untreated, treated with NC medium, or treated 48 h with 250 pM Aβd/t were mapped onto a matrix grid of the hippocampus using fiduciary points to stretch and fit multiple slice data onto a single summary map as previous described [29]. Similar to the localization of rods in response to Aβsyn oligomers [29], a treatment also repeated here (data not shown), the Aβd/t-induced rods were mainly localized to the polymorphic hilar region of the dentate gyrus and along the mossy fiber tract into the CA3 region (Figure 4). Furthermore, the numbers of rods per grid square are on average 2-3 fold higher than for the comparable region of the slices treated with Aβsyn oligomer (not shown, but the maximum rods per square on the hot scale in Figure 4A is 60 compared with the maximum of 15 rods per square for synthetic Aβ-treated slices previously published [29]). Rod numbers in slices treated with the gel filtration fraction of NC medium were not significantly different from untreated controls and thus data from these slices were combined to make the control panel (Figure 4A). Similar to what was observed in dissociated neurons (Figure 1B) pretreatment of Aβd/t with 6E10 antibody reduced the rod numbers in slices to control levels (data not shown).


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)

Numbers of rods induced by Aβd/t are highest in the dentate gyrus and mossy fiber tract. (A) Organotypic hippocampal slices were cultured for at least 8-10 days and were left untreated or treated with 1X Aβd/t, the same amount of the equivalent NC medium fraction, or scrambled Aβ peptide. After 48 h, slices were fixed and immunostained for cofilin and DNA (DAPI), and rods were quantified by counting with a 60x objective. Rod mapping from multiple slices onto a matrix grid of the hippocampus was performed as previously described using fiduciary markers from the stained nuclei layers to align hippocampal regions [29]. There were no differences detected in rod numbers or distribution between the untreated slices and those treated with NC medium or scrambled peptide and these were all combined to give the control panel. Rods induced by the Aβd/t were heavily concentrated over the dentate gyrus and mossy fiber tract (n = 18 for control slices and n = 12 for Aβd/t treated). (B) Rod quantification averaged per field over different regions of the slices. Each field acquired with the 60x objective has about 6-7 matrix grid squares. The only regions of significance (# = p = 0.05) for the rod numbers are in the dentate gyrus and mossy fiber tract.
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Figure 4: Numbers of rods induced by Aβd/t are highest in the dentate gyrus and mossy fiber tract. (A) Organotypic hippocampal slices were cultured for at least 8-10 days and were left untreated or treated with 1X Aβd/t, the same amount of the equivalent NC medium fraction, or scrambled Aβ peptide. After 48 h, slices were fixed and immunostained for cofilin and DNA (DAPI), and rods were quantified by counting with a 60x objective. Rod mapping from multiple slices onto a matrix grid of the hippocampus was performed as previously described using fiduciary markers from the stained nuclei layers to align hippocampal regions [29]. There were no differences detected in rod numbers or distribution between the untreated slices and those treated with NC medium or scrambled peptide and these were all combined to give the control panel. Rods induced by the Aβd/t were heavily concentrated over the dentate gyrus and mossy fiber tract (n = 18 for control slices and n = 12 for Aβd/t treated). (B) Rod quantification averaged per field over different regions of the slices. Each field acquired with the 60x objective has about 6-7 matrix grid squares. The only regions of significance (# = p = 0.05) for the rod numbers are in the dentate gyrus and mossy fiber tract.
Mentions: The numbers of rods in fields taken from multiple organotypic hippocampal slices that were untreated, treated with NC medium, or treated 48 h with 250 pM Aβd/t were mapped onto a matrix grid of the hippocampus using fiduciary points to stretch and fit multiple slice data onto a single summary map as previous described [29]. Similar to the localization of rods in response to Aβsyn oligomers [29], a treatment also repeated here (data not shown), the Aβd/t-induced rods were mainly localized to the polymorphic hilar region of the dentate gyrus and along the mossy fiber tract into the CA3 region (Figure 4). Furthermore, the numbers of rods per grid square are on average 2-3 fold higher than for the comparable region of the slices treated with Aβsyn oligomer (not shown, but the maximum rods per square on the hot scale in Figure 4A is 60 compared with the maximum of 15 rods per square for synthetic Aβ-treated slices previously published [29]). Rod numbers in slices treated with the gel filtration fraction of NC medium were not significantly different from untreated controls and thus data from these slices were combined to make the control panel (Figure 4A). Similar to what was observed in dissociated neurons (Figure 1B) pretreatment of Aβd/t with 6E10 antibody reduced the rod numbers in slices to control levels (data not shown).

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