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, 10 (1), 2369

Bacterial DNA Promotes Tau Aggregation

Affiliations

Bacterial DNA Promotes Tau Aggregation

George Tetz et al. Sci Rep.

Abstract

A hallmark feature of Alzheimer's disease (AD) and other tauopathies is the misfolding, aggregation and cerebral accumulation of tau deposits. Compelling evidence indicates that misfolded tau aggregates are neurotoxic, producing synaptic loss and neuronal damage. Misfolded tau aggregates are able to spread the pathology from cell-to-cell by a prion like seeding mechanism. The factors implicated in the initiation and progression of tau misfolding and aggregation are largely unclear. In this study, we evaluated the effect of DNA extracted from diverse prokaryotic and eukaryotic cells in tau misfolding and aggregation. Our results show that DNA from various, unrelated gram-positive and gram-negative bacteria results in a more pronounced tau misfolding compared to eukaryotic DNA. Interestingly, a higher effect in promoting tau aggregation was observed for DNA extracted from certain bacterial species previously detected in the brain, CSF or oral cavity of patients with AD. Our findings indicate that microbial DNA may play a previously overlooked role in the propagation of tau protein misfolding and AD pathogenesis, providing a new conceptual framework that positions the compromised blood-brain and intestinal barriers as important sources of microbial DNA in the CNS, opening novel opportunities for therapeutic interventions.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Tau seeding aggregation assay. (A) Full-length Tau seeds were prepared by incubating tau monomer (50 µM) with 12.5 µM heparin in 10 mM HEPES pH 7.4, 100 mM NaCl for 5 days at 37 °C with shaking. Aggregation was monitored by ThT fluorescence. (B) Tau aggregates exhibit the typical ThT fluorescence spectrum with a maximum around 495 nm when excited at 435 nm. (C) The aggregation state was further confirmed by sedimentation followed by western blot, showing that the majority of tau appeared in the pellet in the form of large molecular weight bands. (D) the morphological characteristics of the tau aggregates were studied by transmission electron microscopy after negative staining with uranyl acetate. (E) The Tau aggregation assay was performed on 96 well plates using 22 µM Tau monomer, 4.4 µM heparin, 10 µM Thioflavin T, using cyclic agitation (1 min shaking at 500 rpm followed by 29 min without shaking). Aggregation was followed over time by ThT fluorescence using a plate spectrofluorometer (excitation: 435; emmision: 485). Graph show the mean and SD of three replicates. (F) Relationship between the quantity of tau oligomers and the Tau-PMCA signal (time to reach 50% aggregation).
Figure 2
Figure 2
Effect of DNA extracted from diverse sources on tau aggregation. To study the effect of DNA on tau aggregation, monomeric tau (22 µM) under the conditions described in Fig. 1E, was incubated with preparations containing 100 ng of DNA extracted from different bacterial species including Pseudomonas aeruginosa (PA), Tetzosporium hominis (TH), Tetzerella alzheimeri (TA), Escherichia coli ATCC 25922 (EC25), Escherichia coli ATCC 472217 (EC47), Porphyromonas gingivalis (PG), Borrelia burgdorferi (BB). We also incubated tau with same amount of DNA extracted from Candida albicans (CA) and human samples. In all experiments the signal at time zero, corresponding to buffer + DNA + heparin + ThT + monomeric tau was substracted from the values. (A) tau aggregation was monitored over time by ThT fluorescence. Data corresponds to the average ± standard error of experiments done in triplicate (except for control without seeds that was performed in quintuplicate). (B) The lag phase, estimated as the time in which ThT fluorescence was higher than the threshold of 40 arbitrary units, was calculated for each experiment. The points represent the values obtained in each of the replicates. Data was analyzed by one-way ANOVA, followed by Tukey multiple comparison post-test. *P < 0.01; **P < 0.001.
Figure 3
Figure 3
Influence of different concentration of E. coli ATCC 25922 DNA on tau aggregation. To study whether the promoting effect of E. coli DNA can be observed at different concentrations of DNA, we incubated monomeric tau under the conditions described above (Figs. 1E and 2) with 1000, 100 and 10 ng of DNA extracted from E. coli ATCC 25922. (A) tau aggregation was monitored overtime by ThT fluorescence. Data corresponds to the average ± standard error of experiments done in triplicate. (B) The lag phase, estimated as the time in which ThT fluorescence was higher than the threshold of 40 arbitrary units, was calculated for each experiment. The points represent the values obtained in each of the replicates. Data was analyzed by one-way ANOVA, followed by Tukey multiple comparison post-test. *P < 0.01; **P < 0.001.
Figure 4
Figure 4
Dose-dependent effect of DNA from Porphyromonas gingivalis on tau aggregation. Monomeric tau was incubated under the conditions described above (Figs. 1E and 2) with 1000, 100 and 10 ng of DNA extracted from P. gingivalis. Сontrol probes of 1000 ng of P. gingivalis DNA were treated with DNase I to remove DNA from the sample. (A) tau aggregation was monitored overtime by ThT fluorescence. Data corresponds to the average ± standard error of experiments done in triplicate. (B) The lag phase, estimated as the time in which ThT fluorescence was higher than the threshold of 40 arbitrary units, was calculated for each experiment. The points represent the values obtained in each of the replicates. Data was analyzed by one-way ANOVA, followed by Tukey multiple comparison post-test. *P < 0.05; **P < 0.001.
Figure 5
Figure 5
Effect of DNA from Porphyromonas gingivalis on tau aggregation measured by TEM and sedimentation assay. Monomeric tau was incubated under the conditions described above (Figs. 1E, 2 and 4) with 1000 ng of DNA extracted from P. gingivalis. (A) tau aggregation was monitored overtime by ThT fluorescence. Data corresponds to the average ± standard error of experiments done in triplicate. (B) Aliquots taken after 300 h of incubation were taken and loaded into TEM grids and stained with uranyl acetate, as indicated in methods. Scale bar corresponds to 50 nm. (C) The aggregation state was further confirmed by sedimentation followed by dot blot. The left panel shows the signal obtained in the pellet fraction after 300 h of incubation for individual wells or the pool of the samples in the presence and absence of DNA. For this experiment, the pellet was resuspended in 200 µl of the same buffer used for aggregation (10 mM Hepes pH 7.4, 100 mM NaCl) and 2 µl of a 8-fold dilution of this sample was loaded in the membrane. The right panel shows the dot blot signal of distinct concentrations of recombinant monomeric tau. (D) The UV spectra of solubilized pellet was measured between 240 and 300 nm for the pool of replicates incubated alone or in the presence of 1000 ng of PG DNA.
Figure 6
Figure 6
Agarose gel electrophoresis images of DNA. Lane 1, XL 1 kb Plus DNA Marker; Lane 2 DNA P. aeruginosa; Lane 3 DNA T. hominis; Lane 4 DNA T. alzheimeri; Lane 5 DNA C. albicans; Lane 6 DNA E. coli ATCC 472217; Lane 7 DNA E. coli ATCC 25922; Lane 8 Human DNA; Lane 9 DNA B. burgdorferi; Lane 10 DNA P. gingivalis.

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