Alzheimer's disease-type neuronal tau hyperphosphorylation induced by A beta oligomers

Abstract

Alzheimer's disease (AD) is characterized by presence of extracellular fibrillar A beta in amyloid plaques, intraneuronal neurofibrillary tangles consisting of aggregated hyperphosphorylated tau and elevated brain levels of soluble A beta oligomers (ADDLs). A major question is how these disparate facets of AD pathology are mechanistically related. Here we show that, independent of the presence of fibrils, ADDLs stimulate tau phosphorylation in mature cultures of hippocampal neurons and in neuroblastoma cells at epitopes characteristically hyperphosphorylated in AD. A monoclonal antibody that targets ADDLs blocked their attachment to synaptic binding sites and prevented tau hyperphosphorylation. Tau phosphorylation was blocked by the Src family tyrosine kinase inhibitor, 4-amino-5-(4-chlorophenyl)-7(t-butyl)pyrazol(3,4-D)pyramide (PP1), and by the phosphatidylinositol-3-kinase inhibitor LY294002. Significantly, tau hyperphosphorylation was also induced by a soluble aqueous extract containing A beta oligomers from AD brains, but not by an extract from non-AD brains. A beta oligomers have been increasingly implicated as the main neurotoxins in AD, and the current results provide a unifying mechanism in which oligomer activity is directly linked to tau hyperphosphorylation in AD pathology.

Fig. 1

Biochemical characterization and neurotoxicity of ADDLs. A, SDS-PAGE separation followed by Western blot with 6E10 and NU1 shows that ADDLs preparations contain a mixture of monomers, trimers, tetramers and higher molecular weight oligomers. Western blot with 6E10 and NU1 shows that Aβ fibril preparations contain Aβ oligomers, that can only be detected by 6E10 antibody. B, Time-dependent toxicity of ADDLs to hippocampal neurons. Mature hippocampal E-18 cultures (19 days in vitro) were incubated with ADDLs or vehicle for 24, 48 or 96 hours. Values represent means ± SD of the relative absorbance measured using 6 wells for each experimental condition for three independent experiments.

Fig. 2

ADDLs induce tau hyperphosphorylation in B103 neuroblastoma cells B103 neuroblastoma cells (Panels A–D) were maintained for 6 hours at 37 °C in the presence of vehicle (Panels A, B) or 1 µM biotinylated ADDLs (Panels C, D). Panels A and C shows DAPI staining of vehicle (A) or biotinylated ADDLs treated (C) cells. Panels B and D show cells treated with vehicle and biotinylated ADDLs, respectively, for 6 hours and immunostained with AT8 P-tau antibody. A time-course investigation on ADDL-induced tau phosphorylation is shown in Panels E–H. Panels I and J: Primary hippocampal cultures prepared from frozen dissociated rat hippocampal cells (21 days in vitro) were maintained for 6 hours at 37 °C in the presence of vehicle (Panel I) or 1 µM biotinylated ADDLs (Panel J). ADDLs-induced tau phoshorylation (AT8 labeling, panel J arrowheads) is detected in cells that are also labeled with ADDLs (Panel I, arrowheads). Panel K shows a typical image of Thr231 tau immunofluorescence in mature hippocampal neurons treated with 500 nM ADDLs. Double labeling using an ADDL specific antibody (Panel L) reveals that only the neuron that has ADDLs bound shows high P-tau fluorescence (Panel K, M). A cell that does not have ADDL labeling (circled in Panel L and indicated by an arrow in Panel M) also does not have P-tau fluorescence. Panel N shows a typical image of Thr231 tau immunofluorescence in mature hippocampal neurons treated with 10 µM Aβ fibrils. Double labeling using NU1 (Panel O, P) reveals the presence of large extracellular aggregates that are not specifically bound synapses.

Fig. 3

ADDL-induced neuronal tau hyperphosphorylation at Ser404, Thr231 and Thr181 is blocked by NU1 antibody. Primary cultures of E-18 hippocampal neurons (20 days in vitro) were maintained for 4 hours at 37 °C in the presence of vehicle, 500 nM ADDLs, 10 µM Aβ fibrils or 500 nM NU1 + 500 nM ADDLs, as indicated. When present, NU1 antibody was added 30 minutes before addition of ADDLs. Panels A–D, P-Ser404 tau immunofluorescence in mature hippocampal neurons treated with vehicle (Panel A), 500 nM ADDLs (Panel B), 10 µM Aβ fibrils (Panel C) or 500 nM NU1 + 500 nM ADDLs. Panels E–H and K–O, P-Thr231 tau immunofluorescence in mature hippocampal neurons treated with vehicle (Panel E), 500 nM ADDLs (Panel F), 10 µM Aβ fibrils (Panel G or N), 500 nM NU1 + 500 nM ADDLs (Panel H), 500 nM IgG + 500 nM ADDLs (Panel M) or 10 µM Aβ fibrils + 500 nM NU1 (Panel O). Cells were immunostained for tau as described in “Methods”. In order to allow direct comparison of tau imunnofluorescence, identical conditions and exposure times for image acquisition were employed for the different experimental conditions. This explains the faint appearance of the images of control cultures. Scale bar corresponds to 20 µm. Panels I and J show, respectively, quantitative analysis of P-Ser404 and P-Thr231 tau immunofluorescence obtained in at least three separate experiments (60 images analyzed per experimental condition) using Image J software (NIH Windows version). * indicates a statistically significant (p< 0.005) increase compared to control cultures.

Fig. 4

NU1 antiboby prevents ADDL-induced tau hyperphosphorylation. Primary cultures of E-18 hippocampal neurons (20 days in vitro) were maintained for 4 hours at 37 °C in the presence of vehicle, 100 nM ADDLs, 500 nM ADDLs, 10 µM Aβ fibrils or 500 nM NU1 + 500 nM ADDLs as indicated. Panel A, Western blots showing the immunoreactivity for 2 different phosphorylated tau epitopes in hippocampal culture lysates. Identical protein amounts (10 µg) were applied to each lane. As a loading control, cyclophilin B was used to allow direct comparison between the total protein mass applied to different lanes. Panels B–D shows the quantification of the ratio between phosphorylated tau at Ser404/cyclophilin B (Panel B), Thr231/cyclophilin B (Panel C), Thr181/cyclophilin B (Panel D). Bars represent means ± SD values from densitometric analysis of the bands obtained in four separate experiments using Image J software (NIH Windows version). ** indicates a statistically significant (p< 0.007) and ** indicates a statistically significant (p< 0.05) increase compared to control cultures.

Fig. 5

PP1 and LY 294002 block ADDLs-induced tau hyperphosphorylation. Primary cultures of E-18 hippocampal neurons (20 days in vitro) were maintained for 4 hours at 37 °C in the presence of vehicle, 500 nM ADDLs, 500 nM NU1 + 500 nM ADDLs, 10 µM PP1 + 500 nM ADDLs or 5 µM LY 294002 + 500 nM ADDLs. When present, NU1 antibody, PP1 and LY 294002 were added 30 minutes before addition of ADDLs. PP1 and LY 294002 were added again immediately before ADDLs addition. Double labeling using P-Thr231 (green) and NU1 (red) antibodies in neurons treated with vehicle (Panels A and B), 500 nM ADDLs (Panels D and E), 10 µM PP1 + 500 nM ADDLs (Panels G and H), 5 µM LY 294002 + 500 nM ADDLs (Panels J and K) or 500 nM NU1 + 500 nM ADDLs (Panels M and N). Scale bar corresponds to 20 µm. Panel P shows quantitative analysis of P-Thr231 tau immunofluorescence for neurons treated with vehicle, 500 nM ADDLs, 10 µM PP1 + 500 nM ADDLs, 5 µM LY 294002 + 500 nM ADDLs, 500 nM NU1 + 500 nM ADDLs obtained in three separate experiments (60 images analyzed per experimental condition) using Image J software (NIH Windows version). * indicates a statistically significant (p< 0.005) increase compared to control cultures.

Fig. 6

Soluble extract from AD brain containing ADDLs induces neuronal tau hyperphosphorylation in vitro. Primary cultures of E-18 hippocampal neurons (20 days in vitro) were maintained for 3 hours at 37 °C in the presence of aqueous soluble extracts from AD- or non-AD brains. When present, NU1 antibody was added 30 minutes before addition of soluble brain extracts. Double labeling using anti-P-Thr231 (green; Panels A, D and G) and NU1 (red; Panels B, E and H) antibodies are shown for neurons treated with 1 mg/ml non-AD soluble extract (Panels A–C), 1 mg/ml AD soluble extract (Panels D–F), or 1 mg/ml AD soluble extract + 500 nM NU1 (Panels G–I). Merge images of phosphotau and NU1 labeling for neurons treated with non-AD soluble extract and soluble AD extract are shown in Panels C, F and I, respectively. Scale bar corresponds to 20 µm. Panel J shows quantitative analysis of P-Thr231 tau immunofluorescence in neurons treated with 1 mg/ml non-AD soluble extract, 1 mg/ml AD soluble extract or 1 mg/ml AD soluble extract + 500 nM NU1 obtained from three separate experiments (60 images analyzed per experimental condition) using Image J software (NIH Windows version). * indicates a statistically significant (p< 0.02) increase compared to cultures exposed to Non-AD extract. # indicates a statistically significant (p< 0.03) decrease compared to cultures exposed to AD soluble extract.