PubMed: 28528849

Title
Extracellular low-n oligomers of tau cause selective synaptotoxicity without affecting cell viability.
Journal
Alzheimer's & dementia : the journal of the Alzheimer's Association
Volume
13
Issue
None
Pages
1270-1291
Date
2017-11-01
Authors
Chandupatla RR | Kaniyappan S | Mandelkow E | Mandelkow EM

Evidence 92aedf7b59

In our hands, these aggregated tau species formed in different conditions did not show any significant release of LDH when applied on the differentiated SH-SY5Y cells (Supplementary Fig. 8A), and they did not show a significant reduction in cell viability by the MTTassay (Supplementary Fig. 8B).

Evidence 198b8ed8a7

The fibril-treated cells (cross-linked with GA or not) did not reduce the spine density significantly (Fig 4C, bars 8 and 9).

Evidence 19312cedc6

Recently, extracellular tau oligomers were shown to impair long term potentiation (LTP) and memory [40].

Evidence 98bb9dc014

For comparison, in the case of Ab oligomers, only higher concentrations (w1 mM) cause an appreciable spine reduction of w30% (Fig. 4C, bar 3) (Zempel et al., 2010 [30]).

Evidence e52a09492d

We reasoned that microglia and other cell types in the hippocampus might act as mediators for the cytotoxicity caused by TauRDΔK oligomers, which could explain why cytotoxicity was not observed in the cell culture systems.

Evidence 636278b6d4

We indeed found that there is an overexpression (w15%) of Nox1 protein (a component of NADPH oxidase complex) by Western blot, suggesting the role of NADPH oxidase complex as a potential source of ROS

Evidence dc4255b3e1

These findings suggest that the ROS production induced by extracellular TauRDΔK oligomers might cause the activation of NADPH oxidase complex

Evidence 60cd28c964

TauRDΔK comprises the structural elements required for the pathologic assembly of tau filaments, and it causes reversible memory deficits and synapse loss in regulatable transgenic mice [11,25].

Evidence a2ed343c07

Analysis of sarkosyl extracts of brain homogenates of mice expressing the pro-aggregant repeat domain TauRDΔK revealed that oligomers are present, partly in a disulfide–cross-linked form (Supplementary Fig. 1).

Evidence e7532b818f

During early stages of assembly, there is some increase in ThS intensity, combined with a pronounced increase in ANSfluorescence (Fig. 2Aand B), which is due to oligomers, indicating a change in conformation without increase in beta-structure

Evidence 38cf7c2b70

As shown earlier, TauRDΔK-expressing mice display loss of neurons in the CA3 and other regions of the hippocampus [11,24].

Evidence 8f346d206e

By contrast, monomers of TauRDΔK even at 10 mM concentration did not cause any significant ROS increase (Fig. 5B).

Evidence 3510e11a78

By contrast, there was no significant increase in the calcium level in cells treated with TauRDΔK monomers even at higher concentration (10 mM) (Fig. 5E and F, green curves).

Evidence 681bd72ff1

During the transition from oligomers to polymers, the ANS fluorescence remains roughly constant, whereas ThS fluorescence increases strongly (Fig. 2B)

Evidence 91ea9a176c

To address this question, we first applied the oligomers directly after the purification of the protein eluting from the Butyl FF 16/ 10 column (without buffer exchange; 1, 5, and 10 mM) to SHSY5Y cells and observed a variety of toxic effects, including pronounced reduction in the cell viability (by MTT assay, Fig. 3A), increase in apoptotic cells (by Hoechst staining, Supplementary Fig. 4A), loss of mitochondrial membrane potential (by JC1 assay, Supplementary Fig. 4B), caspase 3/7 activation (Supplementary Fig. 4C-D), and cytochrome-c release (Supplementary Fig. 4), within 5 hours of incubation.

Evidence 32181c287b

Consistent with this, NeuN staining of the slices fixed after 48 hours of treatment with TauRDΔK oligomers revealed no reduction in the neuronal number in all regions of the hippocampus (CA1, CA3, and DG) (Fig. 3C and D, Supplementary Fig. 7), confirming that TauRDΔK oligomers do not cause cell death in the OHSC model as well.

Evidence f2db971ceb

There was no significant change in the LDH release in oligomer-treated cells compared with controls indicating that TauRDΔK oligomers do not compromise the membrane integrity (Supplementary Fig. 5G-H).

Evidence 648e367450

In this case, there was a dramatic loss (up to 50%) of spines in TauRDΔK oligomer-treated cells compared with monomer-treated cells (Fig. 4A and B, bar 3).

Evidence d34a9cdeec

Drebrin, a neuronal actin-binding protein involved in spinogenesis and synaptogenesis, was decreased by up to 60% consistent with the reduced number of spines (Fig. 4D, bars 3, 6, and 9).

Evidence 586c269393

Endogenous tau protein retained its normal axonal localization and did not missort into the cell body and dendrites (Fig. 6A8), although there was a reduction in the spine density (Fig. 6B6) in the TauRDΔK oligomer-treated neurons.

Evidence 87b7a2e411

The results revealed that both TauRDΔK and TauFLΔK oligomers reduce the density of spines up to 50% compared with the buffer-treated cells.

Evidence 305c0a3a6b

PSD-95, a marker of postsynaptic spines, decreased up to 50% in the TauRDΔK oligomer-treated cells, compared with buffer- and monomer-treated cells

Evidence 95a97e747f

Similarly, the GluR1 subunits of a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (characteristic of mature spines and necessary for LTP and calcium signaling) decreased in the oligomer-treated samples up to 60%.

Evidence fb65c59803

We also looked at the presynaptic protein synaptophysin, which was not significantly altered in TauRDΔK oligomer-treated cells compared with buffer- or monomer-treated cells (Fig 4D, bar 12). However, at higher concentration, synaptophysin tends to be reduced.

Evidence 5d713c393c

We observed an oligomer-dependent increase in the ROS production in the mature rat primary hippocampal neurons in all cellular compartments (Fig. 5A).

Evidence 9af7c1f38a

However, we did not find significant changes in the expression level of Nox2 protein (another component of NADPH oxidase complex) (Fig. 5C).

Evidence 051fe75527

The ratio of 340 to 380 nm in TauRDΔK oligomer-treated cells showed a steady concentration-dependent increase in the intracellular calcium with a maximum reached at 20 minutes of incubation with oligomers (10 mM) occurring in all cell compartments (Fig. 5D; arrows).

Evidence 85a27ec17b

We did not observe any increase in the phosphorylation of the tau repeat domain (as seen by the antibody 12E8) after treating the neurons with TauRDΔK oligomers.

Evidence 5cddf60302

We also checked the phosphorylation of tau at other sites (e.g., using the antibody AT8, reacting only with the endogenous tau) and did not observe an increase in the phosphorylation.

Evidence 5831b2734b

Tau oligomers from TauRDΔK and TauFLΔK mice reduced the density of the synapses by w50%, whereas tau from wild-type mice had no effect on the density (Fig. 7G).

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