Dual Mechanism of Toxicity for Extracellular Injection of Tau Oligomers versus Monomers in Human Tau Mice

Abstract

The mechanism of tau toxicity is still unclear. Here we report that recombinant tau oligomers and monomers, intraventricularly injected in mice with a pure human tau background, foster tau pathology through different mechanisms. Oligomeric forms of tau alter the conformation of tau in a paired helical filament-like manner. This effect occurs without tau hyperphosphorylation as well as activation of specific kinases, suggesting that oligomers of tau induce tau assembly through a nucleation effect. Monomers, in turn, induce neurodegeneration through a calpain-mediated tau cleavage that leads to accumulation of a 17 kDa neurotoxic peptide and induction of apoptotic cell death.

Keywords: Alzheimer’s disease; hTau mice; paired helical filaments; tau protein.

Fig. 1.

Tau oligomers, but not monomers, induce a conformational change of tau protein. A) Representative western blot of brain extracts from mice injected with control ACSF solution or tau peptides (ICV for 3h) using a conformational tau antibody (MC1) and total tau antibodies (Tau5 as well as Tau46) for detection. Densitometric quantification shows a significant tau pathological conformational change revealed by MC1 antibody induced by oligomers, but not by monomers. B) Representative western blot of insoluble tau fraction by sarkosyl detergent technique extracts from mice injected with control ACSF solution or tau peptides (ICV for 3 h) using a Tau46 antibody for detection. Treatment with oligomeric preparations produced a significant increase in immunoreactivity for a band at approximately 75 kDa molecular weight. mTau, in turn, did not produce any significant change in the 75 kDa band. In all cases an antibody raised against GAPDH served as a loading control. The data are mean ± SEM, *p < 0.05; **p < 0.01 versus control while °p < 0.05 versus mono by one-way ANOVA followed by Bonferroni post hoc test, n = 6.

Fig. 2.

Tau oligomers do not alter the phosphorylation state of tau protein. Representative western blot of brain extracts (20 μg protein) from mice injected with control ACSF solution or tau peptides (ICV for 3 h) using antibodies specific for the detection of four pathological tau phosphorylation sites: AT8, pS396, pS262, and pS422. An antibody raised against GAPDH served as a loading control. Densitometric quantification shows that both oTau and mTau were not able to alter the phosphorylation state of tau. The data are mean ± SEM, n = 6.

Fig. 3.

Tau peptides do not affect the major kinases involved in the regulation of tau protein. Representative western blotting of brain extracts from mice injected with control ACSF solution and tau peptides (ICV for 3h) using CDK5/p35 (A), pGSK3β (B), and pJNK, pP38, and pERK1/2 (C) antibodies for detection. Densitometric quantification shows that the activity of the major kinases involved in the regulation of tau protein is not affected by both oTau and mTau peptides. A) An antibody raised against GAPDH was used as a loading control. B, C) pGSK3β, pJNK, pP38, and pERK1/2 levels were standardized against their respective total protein amount. The data are mean ± SEM, n = 6.

Fig. 4.

Tau monomers trigger calpain-mediated generation of a 17 kDa tau fragment. A) Representative western blotting of brain extracts from mice injected with control ACSF solution or tau peptides (ICV for 3 h) using calpain 1 and spectrin antibodies for detection. Densitometric quantification shows that the levels of the two enzymes are significantly increased by mTau but not oTau. B) Representative western blotting of brain extracts from mice injected with control ACSF solution or tau peptides (ICV for 3 h) using Tau 1 antibody for detection. Only after treatment with monomeric preparations was present a band at approximately 17 kDa molecular weight. In all cases an antibody raised against GAPDH served as a loading control. The data are mean ± SEM, *p < 0.05; **p < 0.01 versus control by one-way ANOVA followed by Bonferroni post hoc test, n = 6.

Fig. 5.

Tau monomers induce apoptotic cell death and maintain their toxicity also after 4 days treatment without affecting tau conformational changes. A) Representative western blotting of brain extracts from mice injected with control ACSF solution and tau peptides (ICV for 3 h) using BAX and Bcl2 antibodies for detection. Densitometric quantification shows that mTau causes a significant increase in proapoptotic protein BAX and a parallel decrease in the antiapoptotic protein Bcl2. By contrast, oTau caused only a significant increase in n Bax protein levels. B) Representative western blotting of brain extracts from mice injected with control ACSF solution and tau peptides (ICV for 4 days) using MC1 and BAX antibodies for detection. mTau does not modify the pathological conformation of Tau and maintains the ability to mediate apoptotic cell death. In all cases an antibody raised against GAPDH served as a loading control. The data are mean ± SEM, *p < 0.05; **p < 0.01 versus control by one-way ANOVA followed by Bonferroni post hoc test, n = 6.