Abstract
BackgroundPreviously 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.ResultsHere 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.ConclusionsTogether these data support a mechanism by which Aβd/t alters the actin cytoskeleton via effects on cofilin in neurons critical to learning and memory.
Highlights
Proteolytic cleavage of amyloid precursor protein (AbPP) by b- and g-secretases gives rise to Ab peptides ranging in length from 36-43 amino acids [1,2,3,4,5,6]
Because an early indication of AD is blockage in axonal transport that leads to axonal swellings [40,41] and synaptic loss [42], we investigated the ability of physiologically relevant amounts of Ab dimer/trimer (Abd/t) to induce cofilin-actin rod formation in rat hippocampal neurons and organotypic slices
The amounts of Ab dimer/trimer (Abd/t) and Ab monomer (Abm) in their respective pooled fractions from gel filtration chromatography (Additional file 1) were determined directly from dot blot assays to be approximately 1.1 ng/mL and 3.6 ng/mL, respectively, equal to about 250 pM and 800 pM. This value for Abd/t concentration is close to that estimated from the Abd/t immunostaining on Western blots after epitope exposure by boiling the membrane [20], and is quite similar to the concentration of Abd/t in 7PA2 medium estimated by Freir et al [24]
Summary
Proteolytic cleavage of amyloid precursor protein (AbPP) by b- and g-secretases gives rise to Ab peptides ranging in length from 36-43 amino acids [1,2,3,4,5,6]. Onset familial AD is linked with high penetrance to mutations that lead to increased production of the most amyloidogenic species, Ab1-42 [4,7,8,9,10]. The term Ab is used to describe a spectrum of peptide species, the effects of different Ab peptide species on neuronal function or morphology are not the same [12,13]. Unlike synthetic Ab peptide oligomers or fibrils, which generally are used at μM or greater concentrations, ADDLs are toxic to cultured neurons at nanomolar concentrations [15] and at 500 nM they prevent high frequency stimulation-induced long-term potentiation (LTP) [16]. We reported 1 μM synthetic human amyloid beta oligomers induced cofilin dephosphorylation (activation) and formation of cofilin-actin rods within rat hippocampal neurons primarily localized to the dentate gyrus
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