PubMed: 17403556

Alzheimer's disease-type neuronal tau hyperphosphorylation induced by A beta oligomers.
Neurobiology of aging
De Felice FG | Klein WL | Acton PJ | Bigio EH | Chen-Dodson E | Fernandez SJ | Jerecic J | Kinney GG | Lacor PN | Lambert MP | Shughrue PJ | Velasco PT | Wu D

Evidence 96076d0d14

Interestingly, we found that tau hyperphosphorylation at Thr231 was completely 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 (Fig. 5).

Evidence 7fff0cc9c0

Inhibition occurred even though ADDLs were still bound to cell surfaces, indicating that those kinases are involved in signal transduction coupling between ADDL binding and tau hyperphosphorylation.

Evidence acbfe086e8

Moreover, the presence of large extracellular aggregates in NU1-treated cultures (Fig. 5N) suggests that the antibody effectively sequesters ADDLs and prevents their interactions with neurons (Fig. 5O). No inhibition of ADDL binding was associated with PP1 and LY294002 (Fig. 5H, I, K and L, respectively), but both kinase inhibitors effectively blocked ADDL-induced tau hyperphosphorylation (Fig. 5G and J).

Evidence 3887705b41

NU1 completely blocked the increase in P-Ser404 and P-Thr231 phosphotau levels induced by ADDLs (Fig. 3D–H).

Evidence 4d08d5bce6

Importantly, pre-incubation of AD brain extracts with NU1 significantly blocked the increase in Thr231 phosphotau immunofluorescence (Fig. 6G), establishing the tau hyperphosphorylation was induced by Abeta oligomers in the AD brain extracts. NU1 also prevented the binding of brain-derived ADDLs to synaptic hot-spots (Fig. 6H and I). In NU1-treated cultures, the presence of large extracellular aggregates indicates that the antibody sequesters ADDLs and prevents their interactions with neurons (Fig. 6I).

Evidence 0b57d99cf1

At longer incubation times (48 and 96 h) ADDLs caused a progressive decrease in MTT reduction (Fig. 1B), which can be due to altered trafficking (Liu et al., 1998) as well as cell death.

Evidence f67d396f06

While vehicle treated cells (Fig. 2A and B) exhibited low phosphotau immunofluorescence, cells treated with 1 microM biotinylated ADDLs (bADDLs) for 6 h (Fig. 2C and D) showed a significant increase in P-tau immunofluorescence (Fig. 2D).

Evidence afb08fca35

We then investigated whether bADDLs also induced tau phosphorylation in rat hippocampal neurons. To this end, we initially used frozen dissociated rat hippocampal cell preparations (Cambrex). Cells were maintained in the presence of 1M bADDLs (Fig. 2I and J) or vehicle (not shown) for 6 h at 37 ◦C. We observed a marked P-tau immunostaining in a subpopulation of neurons that also had ADDLs bound (Fig. 2I, arrowheads), while cells that did not bind bADDLs had no AT8 staining (Fig. 2J).

Evidence a2a4de65d4

Following exposure to ADDLs, double-label immunofluorescence microscopy showed high levels of tau phosphorylated at Thr231, which discriminates among AD and non-AD subjects and patients with other forms of dementia (Hampel et al., 2004, 2003), in neurons with prominent dendritic ADDL binding (detected with NU1, Fig. 2K–M). ADDL binding to synaptic hot-spots in hippocampal neurons is evident in images at highermagnification (60×objective, PanelsLand M).

Evidence 8deaf91293

Vehicle-treated neurons (Fig. 3A and E) exhibited very low phosphotau immunofluorescence, but neurons treated for 4 h with 500nM ADDLs showed significantly higher levels in immunofluorescence of P-Ser404 and P-Thr231 tau (Fig. 3B and F, respectively). Neurons treated for 4 h with 10M Abeta fibrils also showed an increase in immunofluorescence of P-Ser404 and P-Thr231 tau (Fig. 3C and G, respectively).

Evidence ff8da3901c

Verification of the findings from immunofluorescence microscopy was provided by Western blot analysis of hippocampal neuronal lysates with P404, P231 and P181 antiphosphotau antibodies. A 4 h exposure to 500nM ADDLs resulted in a significant increase in tau phosphorylated at the three epitopes, to levels similar to those observed after exposure to 10 M Abeta fibrils (Fig. 4A–D).

Evidence 53c57af8dd

Hippocampal neurons exposed to Abeta fibrils rather than oligomers also showed elevated P-tau immunofluorescence (Fig. 2N–P) However, although fibrils could be seen attached to neurons, they did not bind in the synaptic pattern observed for ADDLs (Fig. 2O and P).

Evidence f27c7500c5

These results confirm that the tau hyperphosphorylation stimulated by soluble ADDL preparations is indeed oligomer-induced. Tau hyperphosphorylation induced by 10M Abeta fibrils (Fig. 3N) was partially blocked (Fig. 3O), consistent with shared epitopes between oligomers and fibrils.

Evidence eac936e5c1

AD brains could induce AD-type tau hyperphosphorylation. Consistent with the results obtained with synthetic ADDLs, we found that treatment of mature hippocampal neuronal cultures with a soluble AD brain extract led to a significant increase in P231 tau phosphorylation (Fig. 6D) compared to cultures treated with a non-AD brain extract (Fig. 6A).

Evidence a5618765cf

We note that these observed increases in tau phosphorylation occurred well before neuronal death (Fig. 1B), consistent with this pathology representing an early stage in neurodegeneration.


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