Extracellular Vesicles Containing P301L Mutant Tau Accelerate Pathological Tau Phosphorylation and Oligomer Formation but Do Not Seed Mature Neurofibrillary Tangles in ALZ17 Mice

Experimental design

Whole brain lysates and EVs were isolated from 4–6 month old wild-type and rTg4510 mice as described previously [19]. Each experimental group contained five ALZ17 mice of mixed gender. Three non-injected controls (NIC) were used for comparison (Fig. 1A). In the figures, ‘exosomes’ is used to indicate EVs. Samples were concentration-matched to 1μg/μl, and 2.5μl were injected bilaterally into the hippocampus (Bregma co-ordinates: anterior/posterior, –2.5 mm; medial/lateral±2.0 mm; dorsal/ventral, –1.8 mm) of 3 month old ALZ17 mice and aged for 6 months (Fig. 1A). Tau pathology was assessed using the phospho-specific AT8 antibody (reactive with tau phosphorylated at S202 and T205), which targets a pathological phosphorylation epitope. Phosphorylation was quantified as percentage area occupied in the hippocampus and as AT8-positive cell counts, and was performed blinded to the experimental condition. Early aggregation events were probed for using the T22 antibody, which is specific to an oligomeric confirmation [20], and mature NFT formation was investigated using the traditional silver staining techniques Gallyas and Bielschowsky. As positive controls, aged Tau58 mice carrying the FTD P301S mutation were used that have well characterized NFT formation [21, 22]. Western blotting of sucrose fractionations to isolate the EVs used in this study is consistent with previously published work [19], confirming that fraction 3 (F3) is the most enriched in both exosome markers and tau (Fig. 1B).

Preparation of the lysate and the extracellular vesicle (EV) fractions

Brains were dissected and the cerebellum and olfactory bulbs were removed. For whole brain lysate, brains were dounce homogenized in 10% w/v phosphate-buffered saline (PBS) containing 1x Complete protease inhibitors (Roche), 25 mM NaF and 20 mM NaVO₄, and then briefly sonicated (6×10 s at 50% amplitude). Brain lysates were centrifuged at 3,000 g for 10 min at 4°C. Supernatants were collected and stored at –80°C until use.

For vesicle fractions, the tissue was processed following an established protocol [18] with minor modifications as described [19]. Briefly, to prepare the vesicle fractions, following dissection, the tissue was manually chopped and incubated in 7 ml of 20 units/ml papain (LS003119, Worthington) in Hibernate-A for 20 min at 37°C. To stop the reaction, 14 ml of ice-cold Hibernate-A with 1x Complete protease inhibitor (Roche), 50 mM NaF, 200 nM Na3VO4, and 10 nM E-64 inhibitor (E3132, Sigma) was added to the tissue, keeping the tissue at 4°C throughout the subsequent steps. The tissue was then disrupted by pipetting up and down with a 10 ml pipette. Cell contamination was cleared by centrifugation at 300 g for 10 min and discarding the pellet. The supernatant was then cleared of large membranous debris by centrifugation at 2,000 g for 10 min and discarding the pellet. For EV purification, the 10,000 g supernatant was syringe filtered at 0.22μm and centrifuged at 100,000 g for 70 min to pellet EVs. The 100,000 g pellet containing EVs plus contaminating proteins was resuspended in 2 ml of 0.95 M sucrose in 20 mM HEPES (15630-080, Life Technologies) before addition to a sucrose-step gradient column. For EV purification, the column consisted of 6×2 ml fraction running from the top 0.25 M, 0.6 M, 0.95 M, 1.3 M, 1.65 M, and 2.0 M at the bottom. Similarly, sucrose step gradients were centrifuged for 16 h at 200,000 g, after which the six fractions were collected. EVs settled typically at 0.95 M sucrose (fraction 3). The protein content for both lysates and vesicle fractions was quantified using the DC protein assay (Bio-Rad). To concentration match lysates and EVs, the lysates were diluted to equal EV fractions at 1μg/ml.

Western blotting

For western blotting, 20μg of protein from brain-derived EV fractions and whole cell extracts were separated by 10% SDS-PAGE electrophoresis and transferred onto PVDF membranes by the Trans-Blot Turbo transfer system (Bio-Rad). Membranes were blocked for 1 h at room temperature in Odyssey blocking buffer (Li-cor) and incubated overnight in primary antibodies. Antibodies used to probe the EV fractions included anti-total tau Tau5 (1:1,000, Millipore), AIP1/Alix (1:1,000, Millipore), and Flot1 (1:500, Santa Cruz). Membranes were washed 3 x in Tris-buffered saline/0.1% Tween-20 (TBST) followed by incubation with a HRP-coupled secondary antibody (1:10,000) for 1 h at room temperature. Membranes were washed a final 3 x in TBST and imaged on the Odyssey Fc imaging system (Li-Cor).

Stereotaxic surgery

Mice were anaesthetized with isoflurane vaporized in oxygen. The head was shaved and depilated before being placed securely in ear bars. Samples were concentration-matched to 1μg/μl, and 2.5μl of the sample was injected bilaterally into the hippocampus (Bregma co-ordinates: anterior/posterior, –2.5 mm; medial/lateral±2.0 mm; dorsal/ventral, –1.8 mm) of 3-month-old ALZ17 mice at an infusion rate of 0.25μl/min, after which mice were aged for 6 months. The same volume of the dye Evans Blue was injected in the same manner to confirm the injection site. For histology, mice were anaesthetized with a lethal dose of pentabarbitone before transcardial perfusion with 30 ml PBS followed by 30 ml 4% paraformaldehyde. Brains were removed from the skull and post-fixed overnight at 4°C.

Histology

Histology and neuropathological quantification of tau phosphorylation were carried out as previously described [23], and all quantification was conducted blinded to the treatment. Briefly, using ImageJ software, the hippocampus was selected as a region of interest and immunoreactivity was quantified as a percentage area. The threshold for positive labeling was determined using the normal distribution of immunoreactivity from the control group and set at three standard deviations from the mean. AT8-positive cell counts were performed in the CA1 region from 2 sections per animal, 14μm apart, at the level of the injection site. AT8 (Millipore) was used at 1:500 dilution, T22 (Millipore) was used at 1:1,000 for immunohistochemistry and 1:500 for fluorescence, Tau5 (Thermo Fisher Scientific) was used at 1:2,000 for immunohistochemistry and 1:1,000 for fluorescence, and fluorescence analyses with AT8 and human tau-specific HT7 (Thermo Fisher Scientific, 1:1,000) were performed as previously described [21]. Gallyas and Bielschowksy silver staining were performed following well-established methods with aged P301S mutant tau transgenic Tau58 mice being used as a positive control for NFT formation [21, 22].

Transgenic EVs increase pathological tau phosphorylation in the hippocampus

To establish whether EVs isolated from rTg4510 mice can accelerate tauopathy, we investigated hyperphosphorylation of tau at the pathogenic phospho-epitope AT8 (Fig. 2A). The percentage area occupied by AT8-immunoreactivity in the hippocampus was significantly increased following injection with transgenic lysate (Fig. 2Avi). This was also the case for rTg4510 EVs, although the response was more variable. In age-matched NIC, AT8-positivity was observed in both the dentate gyrus and the CA3 region of the hippocampus. To control for variation in these regions, the CA1 region as the target of the injection was assessed specifically by calculating the number of phospho-tau-positive cells within this cell layer (Fig. 2B). Both the rTg4510 lysate and rTg4510 EVs significantly increased the number of phospho-tau-positive neurons in the CA1 region (Fig. 2Bvi). No significant differences were observed in hippocampal area, CA1 area or number of CA1 nuclei between treatment groups, indicating comparable sample populations within the dataset with no significant toxicity in the CA1 region as a result of the injections (Fig. 2Bvii-ix). In addition, there was no change in total tau levels in the hippocampus, as probed for by Tau5, an antibody capable of picking up both mouse and human tau in a phosphorylation-independent manner (Fig. 3).

Transgenic EVs seed aggregation of endogenous tau into a soluble oligomeric state

To address whether rTg4510 EVs are able to increase pre-NFT tau species, we used the oligomeric antibody T22 to detect soluble tau aggregates [20]. Although no immunoreactivity was observed in the CA1 layer, punctate signals were observed in clusters throughout the stratum radiatum, a region of high synaptic connectivity harboring Schaffer collateral fibers from the CA3 to CA1 region (Fig. 4A). The number and size of these T22-positive clusters appeared to increase in both the transgenic lysate- and transgenic EV-injected groups (Fig. 4Aii,v). However, this signal co-localized with neither AT8 nor the human transgenic tau, as detected by the HT7 antibody (Fig. 4Aiii,vi). Interestingly, while HT7 did not show clustered immunoreactivity reminiscent of T22, Tau5, an antibody that detects both mouse and human tau, showed clustered immunoreactivity in a pattern similar to T22, that was restricted predominantly in the stratum radiatum (Fig. 4B). Co-labeling confirmed robust and consistent co-localization of T22 and Tau5 puncta (Fig. 4C), suggesting that the oligomeric tau aggregates seeded by rTg4510 lysate and EVs are composed of endogenous murine tau.

Mature NFTs are not seeded by the transgenic lysate or transgenic EVs

To further explore whether the observed hyperphosphorylation reflected mature NFT formation, we probed the lysate- and EV-injected mice with the Gallyas and Bielschowsky silver staining reagents. However, no NFT-like signal was observed across the treatment groups with either technique (Fig. 5A, B), whereas Tau58 mice that were included as a positive control displayed clear NFTs (Fig. 5Avi-vi, Fig. 2Bvi-vi). This suggests that neither the transgenic lysate nor transgenic EVs seeded mature NFT formation under our treatment conditions. As the injected volume was relatively large, we may have experienced a spill-over through the hippocampus and therefore seeding could potentially have occurred elsewhere in the brain. To investigate an exposure to the injected seeds in the hippocampus, an identical volume of the dye Evans blue was injected following the above experimental parameters. The dye was observed in the CA1 region of the hippocampus but also extended both anteriorly and posteriorly to the injection site (Fig. 5C). To be entirely confident that no seeding had taken place elsewhere, we probed for NFTs throughout the hippocampal structure in areas anterior and posterior to the injection site. However, we did not see any evidence of NFT formation as determined by the silver Gallyas impregnation in any of the treatment groups including the rTg4510 lysate (Fig. 5C).