Tau oligomers impair memory and induce synaptic and mitochondrial dysfunction in wild-type mice

Abstract

Background: The correlation between neurofibrillary tangles of tau and disease progression in the brains of Alzheimer's disease (AD) patients remains an area of contention. Innovative data are emerging from biochemical, cell-based and transgenic mouse studies that suggest that tau oligomers, a pre-filament form of tau, may be the most toxic and pathologically significant tau aggregate.

Results: Here we report that oligomers of recombinant full-length human tau protein are neurotoxic in vivo after subcortical stereotaxic injection into mice. Tau oligomers impaired memory consolidation, whereas tau fibrils and monomers did not. Additionally, tau oligomers induced synaptic dysfunction by reducing the levels of synaptic vesicle-associated proteins synaptophysin and septin-11. Tau oligomers produced mitochondrial dysfunction by decreasing the levels of NADH-ubiquinone oxidoreductase (electron transport chain complex I), and activated caspase-9, which is related to the apoptotic mitochondrial pathway.

Conclusions: This study identifies tau oligomers as an acutely toxic tau species in vivo, and suggests that tau oligomers induce neurodegeneration by affecting mitochondrial and synaptic function, both of which are early hallmarks in AD and other tauopathies. These results open new avenues for neuroprotective intervention strategies of tauopathies by targeting tau oligomers.

Figure 1

Experimental design and characterization of tau samples. (A) Mice were divided into four groups, one injected with tau oligomers and PBS, a second group injected with tau fibrils and PBS a third group injected with tau monomers and PBS, and a fourth group injected with tau heparin fibrils and PBS used as a control (*). (B) Schematic representation of a coronal section of a mouse brain with * indicating the injection sites of the tau preparations in PBS. (C) FPLC profile of tau oligomers injected into mice brains. (D) AFM image showing homogeneous population of tau oligomers (scale bar = 140 nm). (E) AFM image of tau fibrils (scale bar = 70 nm). (F) AFM images of tau fibrils prepared with heparin (scale bar = 50 nm).

Figure 2

Tau oligomers impaired the recognition memory in mice. (A) Effects of tau oligomers, fibrils, and monomers on memory were investigated in C57BL/6 mice by using the object recognition task. Histograms indicate the percentage of exploration of the familiar and novel objects. Mice injected with monomers or fibrils spent significantly more time investigating the novel object versus mice injected with tau oligomers, indicating impaired memory, as shown by their inability to recognize the familiar object. #p < 0.01 vs. novel object. (B) Histograms show the corresponding discrimination index (mean ± SD) for the data shown in A. *p < 0.01. As a control, behavioral analyses were performed on mice without injection.

Figure 3

In vivo neurotoxicity of tau oligomers. A-D) Nuclear staining (Hematoxylin) of the hippocampal region CA1 from the hemispheres of mice injected with tau preparations or PBS (Scale bar = 20 μm). E-H) Neuronal staining using the antibody NeuN show neuronal damage in the area injected with tau oligomers (E) in comparison with the other hemispheres injected with fibrils (F) monomers (G) and PBS (H) (scale bar = 10 μm). (I-L) Higher toxicity of tau oligomers versus fibrils, monomers and PBS. Neurodegenerative pyramidal cells in the CA1 hippocampal region were stained with Fluoro-Jade B, emitting green fluorescence (scale bar = 10 μm). M) Number of Fluoro-Jade B positive cells/Total number of cells in the CA1 region, 63% of cells showed signs of damage in the group injected with tau oligomers, compared to less than 6.5% in the group injected with tau fibrils. Data are represented as the mean ± SE. *p < 0.01.

Figure 4

Tau oligomers induce synaptic dysfunction. (A) Representative Western blot of mouse hippocampus homogenate. The levels of synaptophysin, synapsin-1, and septin-11 were measured by band quantification and normalized with the levels of tubulin. PBSo indicates representative bands of hippocampal area injected with PBS in mice also injected with tau oligomers, PBSf indicates PBS injection in mice also injected with tau fibrils, and PBSm indicates PBS injection in mice also injected with tau monomers. (B) Synaptophysin levels were significantly lower in the hemispheres injected with tau oligomers in comparison with the ones injected with fibrils, monomers, or PBS. (C) No significant differences in the levels of synapsin-1 were observed. (D) Only the hemisphere injected with tau oligomers presents a decrease in the level of septin-11. Data are represented as the mean ± SE. *p < 0.01, n = 6

Figure 5

Decrease in synaptophysin in mice injected with tau oligomers. Brain sections were stained with antibody to the presynaptic marker synaptophysin (green) nuclear staining with DAPI (blue). All images were taken from the CA1 region. Hemispheres injected with tau oligomers exhibited a decreased in synaptophysin (A). No decrease in the signal was observed in the hemispheres injected with fibrils (B), monomers (C), and PBS (D). (Scale bars = 10 μm.)

Figure 6

Tau oligomers induce mitochondrial alterations. (A) Representative western blot of mouse brain homogenate. The levels of complex I, complex V, and caspase-9 were measured by band quantification and normalized with the levels of tubulin. Abbreviations are as in Fig. 4. (B) Complex I levels were significantly lower in the hemisphere injected with tau oligomers in comparison with the ones injected with fibrils or monomers. (C) No differences in the levels of complex V were observed in any of the groups. (D) Caspase-9 activation was significantly higher in tau oligomer groups over both tau fibril- and monomer-injected groups. (E-G) Double staining between human tau-specific antibody HT7 (green fluorescence) and mitochondrial porin antibody (red fluorescence) demonstrates the internalization of tau oligomers in CA1 cells and their interaction with the mitochondria. (H) The co-localization of tau oligomers with mitochondria was confirmed by ICA of the signal. Data are represented as mean ± SE. *p < 0.01, n = 6.

Figure 7

Caspase-9 activation in hemisphere injected with tau oligomers. Immunofluorescence using an antibody for pro-caspase-9 was performed. (A and B) Caspase-9 activation (green fluorescence) was observed in hemispheres injected with tau oligomers (A) or fibrils (B). However, the levels of activation were significantly higher in the hemispheres injected with oligomers compared with fibrils, which is not significant. (C and D). No activation of caspase-9 was observed in the hemispheres injected with tau monomers (C) or PBS (D). Nucleus was stained with DAPI (blue). (Scale bars = 10 μm.).