Soluble amyloid beta-protein dimers isolated from Alzheimer cortex directly induce Tau hyperphosphorylation and neuritic degeneration

Abstract

Alzheimer disease is a major cause of cognitive failure, and a pathogenically related but more subtle process accounts for many cases of mild memory symptoms in older humans. Insoluble fibrillar plaques of amyloid β-proteins (Aβ) and neurofibrillary deposits of hyperphosphorylated tau proteins are the diagnostic lesions of AD, but their temporal mechanistic relationship has long been debated. The recent recognition that small, diffusible oligomers may be the principal bioactive form of Aβ raises the key question of whether these are sufficient to initiate cytoskeletal change and neurite degeneration. A few studies have examined the effects of oligomers of synthetic Aβ peptides of one defined length at supraphysiological concentrations, but the existence of such assemblies in the AD brain is not established. Here, we isolated Aβ dimers, the most abundant form of soluble oligomer detectable in the human brain, from the cortices of typical AD subjects and found that at subnanomolar concentrations, they first induced hyperphosphorylation of tau at AD-relevant epitopes in hippocampal neurons and then disrupted the microtubule cytoskeleton and caused neuritic degeneration, all in the absence of amyloid fibrils. Application of pure, synthetic dimers confirmed the effects of the natural AD dimers, although the former were far less potent. Knocking down endogenous tau fully prevented the neuritic changes, whereas overexpressing human tau accelerated them. Coadministering Aβ N-terminal antibodies neutralized the cytoskeletal disruption. We conclude that natural dimers isolated from the AD brain are sufficient to potently induce AD-type tau phosphorylation and then neuritic dystrophy, but passive immunotherapy mitigates this.

Fig. 1.

Cytoskeletal abnormalities induced in primary hippocampal neurons by soluble Aβ oligomers isolated by SEC from the AD cerebral cortex. (A) AD-TBS (Right) or Cont-TBS (Left) was immunoprecipitated with 3D6 (3 μg/mL), eluted with sample buffer containing 4% LDS, and subjected to SEC. SEC of the immunoprecipitaton of AD-TBS resolves Aβ dimers (fractions 8–9) from monomers (fractions 10–11), as detected by Western blot with 6E10 + 2G3 + 21F12. IP, 3D6 immunoprecipitates of the starting Cont-TBS or AD-TBS extracts used for this SEC. (B) Confocal images showing the tau (green) and Tubulin (red) immunoreactivities of the cytoskeleton of hippocampal neurons [days in vitro (DIV) 21] after 3-d treatment with Aβ monomers (SEC fractions 10–11) or Aβ dimers (SEC fractions 8–9) isolated from AD-TBS or the corresponding fractions from Cont-TBS. (Scale bar, 50 μm.) (C) Histograms represent the average number of tau-positive beads along 100-μm lengths of Tubulin-positive neurites under different conditions. Asterisk indicates data significantly different from those of neurons without treatment (P < 0.01 by Student t test). Error bars, SEM. (D) After a 3-d incubation on primary neurons, the conditioned media were immunoprecipitated with Aβ antiserum AW7. Aβ monomers and dimers were precipitated from medium that contained the reconstituted IP-SEC fractions of AD-TBS but not from that with Cont-TBS.

Fig. 2.

Disruption of the neuritic cytoskeleton by soluble Aβ oligomers is dependent on tau expression. (A) Representative Western blots showing the expression of endogenous tau in primary hippocampal neurons (DIV18) transduced with lentivirus encoding EGFP or scrambled RNAi (Cont-RNAi) or RNAi against rat tau (Tau-RNAi-1, Tau-RNAi-2). Western blotting of Tubulin or GFP served as a control. Histograms represent the average expression level of tau, normalized to values in parallel cultures without lentiviral transduction. Asterisks indicate data significantly different from those of neurons without transducton (P < 0.01 by paired Student t test). Data are from five independent experiments; error bars, SEM. (B) Confocal images showing the tau (green) and microtubule (red) cytoskeleton of primary hippocampal neurons (DIV21) transduced with lentivirus encoding Cont-RNAi or Tau-RNAi-1 or Tau-RNAi-2 after 3-d treatment with AβD from AD-TBS or pure Aβ40 S26C (500 nM). (Scale bar, 50 μm.)

Fig. 3.

The neuritotoxic effect of soluble Aβ oligomers is accelerated by expressing human tau. (A) Confocal images (Left) and Western blotting (Right) show the expression of hTau-EYFP in primary hippocampal neurons (DIV7) after transduction with lentivirus encoding hTau-EYFP. (B) Confocal images show the GFP fluorescence (green), microtubule (red) and total tau (blue) cytoskeleton of hippocampal neurons (DIV20 or DIV21) transduced with lentivirus encoding EGFP or hTau-EYFP after 2 or 3 d treatment with AβM or AβD from AD-TBS. (Scale bars, 50 μm.)

Fig. 4.

Alteration of the phosphorylation state of tau at AD-relevant epitopes by soluble Aβ oligomers. Primary hippocampal neurons (DIV19) transduced with lentivirus encoding hTau-EYFP were treated under different conditions, as indicated in the key. Histograms represent the average levels of phosphorylation of human (h) or rat (r) tau at specific epitopes (AT8: Ser202/Ser205; 12E8: Ser262; AT270: Thr181; AT180: Ser231/Thr235; PHF-1: Ser396), normalized to the values in parallel cultures without treatment. Data are means of three independent experiments. Asterisks indicate data significantly different from those of neurons without treatment (P < 0.05 by paired Student t test). Error bars, SEM.

Fig. 5.

Immunological neutralization of the cytoskeletal alterations induced by human Aβ dimers. (A) Confocal images showing the tau (green) and microtubule (red) cytoskeleton of primary hippocampal neurons (DIV21) after 3 d treatment with AβM or AβD from AD-TBS with or without monoclonal antibodies (3D6, 82E1, or 21F12) against human Aβ (each at 3 μg/mL). (Scale bar, 50 μm.) (B) Histograms represent the average number of tau-positive beads along 100-μm lengths of Tubulin-positive neurites under different conditions. Asterisks indicate data significantly different from those of neurons without treatment (P < 0.01 by Student t test). Error bars, SEM. (C) Pull-down of the Aβ dimers by PGA beads added to the conditioned media of the neurons after the 3-d treatment, as detected by Western blot with 6E10 + 2G3 + 21F12.