Dural lymphatics regulate clearance of extracellular tau from the CNS

Initial breeding pairs of K14-VEGFR3-Ig mice [47] were obtained from Dr. Kari Alitalo at the University of Helsinki and the colony was further expanded and maintained at Washington University School of Medicine. The transgenic mice are crossed with C57/BL6 mice. WT and TG mice used in all the experiments were littermates.

WT and K14-VEGFR3-Ig mice were anesthetized with ketamine/xylazine cocktail before being placed in the stereotactic surgery frame. Intrahippocampal injection of recombinant monomeric human tau (labeled and un-labeled) and human serum albumin (HSA) was performed at the following site: bregma − 2.5 mm, lateral 2 mm from midline, 2 mm ventral to the dura. All procedures were performed following protocols approved by the Animal Studies Committee and Washington University School of Medicine.

Recombinant monomeric human tau (1N4R, tau-412, rPeptide) and HSA were conjugated with near-infrared dye cypate in the following manner: Cypate conjugation to proteins was carried out by NHS ester chemistry [48]. Cypate-NHS was prepared by dissolving 31.3 mg (0.05 mmol) cypate in 200 μl DMSO. Next a solution of 5.8 mg (0.05 mmol) NHS, and 4.8 mg (0.025 mmol) EDC was prepared in another 200 μl DMSO solution. Finally the two solutions were mixed and incubated overnight at room temperature. For conjugation, 100 μg tau was reconstituted with 90 μl pH 7.4 1 X PBS buffer. Then 0.75 μl Cypate-NHS was diluted with DMSO into 10 μl, and then added to the 90 μl tau solution and allowed to incubate for 2 h at room temperature.

For HSA conjugation 1.0 mg HSA was dissolved in 200 μl pH 7.4 1 X PBS buffer. Next 1.5 μl, Cypate-NHS DMSO solution was diluted with DMSO into 20 μl, added to the 200 μl HSA PBS solution, and then incubated at room temperature for overnight. Reaction mixtures were dialyzed with 1 X PBS, pH 7.4 at 4 °C for overnight, and then lyophilized. Dye and protein concentrations were determined by protein assay (Bio-Rad) and UV-Vis absorption spectrum, to give dye to protein molar ratio.

In vivo FMT was performed on a Perkin Elmer FMT4000 imaging system. Following intra-hippocampal injection of cypate labeled tau, mice were anesthetized with 2% isoflurane for imaging. Both WT and K14-VEGFR3-Ig mice were imaged at 1, 2, 24, 48, 72 and 168 h post-injection of tau. A 2 μl of solution (1.1 μg) of monomeric human tau or HSA was injected at a flow-rate of 0.2 μl/min. Fluorescence was quantified based on instrument calibration with cypate phantoms. Total fluorescence of tau and HSA fluorescent conjugates was measured in manually drawn ROIs approximating the brain and lymph nodes. To control for variability caused by injection, data for each mouse at each time point were normalized to the fluorescent signal at the 1 h time point.

For plasma measurement, 50 mg/kg of anti-human antibody HJ 8.5 was injected intraperitoneally as described in [44]. Mice were injected with 2 μl of solution (1.1 μg) of unlabeled, monomeric tau 1 h post-antibody administration. Blood was collected at 2, 24, 48, 72, 168 h post-tau injection. Samples were spun at 8000 g for 10 min to obtain plasma. The Simoa HD-1 analyzer (Quanterix Corp) was used to measure human tau in plasma as previously described [44].

Mice were anesthetized by intraperitoneal injection of pentobarbital (200 mg/kg) for perfusion with ice-cold Dulbecco’s PBS with Heparin. Following perfusion, the skull cap was carefully removed and fixed overnight in 4% paraformaldehyde. Intact meninges were then peeled off and stored as floating sections in PBS until immunohistochemistry. For LYVE1 staining, meninges were washed in PBS-Triton X-100 (0.5%) and blocked in PBS-T with 0.5% BSA at room temperature. Sections were incubated with LYVE1-e660 (ThermoFisher) at 1:200 overnight at 4⁰ C. Sections were washed in PBS-T prior to mounting on slides in Prolong Gold antifade reagent with DAPI (ThermoFisher) mounting medium. Slides were imaged on Zeiss Axio Imager Z2 fluorescence microscope and Cytation 5 imaging reader (Biotek Imaging, Inc.). Images were processed on ZEN software suite (Carl Zeiss, Inc.) and Cytation 5 imaging reader.

Bulk of data plotting and statistical analysis were done on GraphPad Prism 5. For tau-cypate (n = 6–9) and HSA-cypate (n = 4–6) injection and brain retention studies, a two-way ANOVA with a Bonferroni post-test by was used to analyze the two groups of mice at each time point (i.e. 2, 24, 48, 72 and 168 h). For plasma tau measurement a mixed effects linear model was used to compare plasma tau (pg/ml) for the two groups (WT n = 5, K14-VEGFR3-Ig n = 6) across five times (2, 24, 48, 72, and 168 h). Akaike’s AIC was used to evaluate 15 ovariance structures to determine the best fit model for the two analyses. Least square estimates of the differences between groups (i.e., mouse types or drug treatment) at each time period were used to compare the trajectory of response over time. All tests were conducted at the alpha = 0.05 level of significance. All tests were run in PROC MIXED of SAS 9.4.

We utilized the K14-VEGFR3-Ig transgenic mice for our studies. These mice lack a functional dural lymphatic system [32] and thus provide a model to study the contribution of this system to clearance of substances from the brain. We first confirmed that these mice lack dural lymphatics by immunohistochemistry for LYVE1, a lymph endothelial cell marker (Fig. 1). WT mice show clear distribution of lymphatic vessels along superior sagittal and transverse sinuses, while K14-VEGFR3-Ig mice have no LYVE1 staining, indicating a complete lack of lymphatic vessels.

To assess clearance of extracellular tau from the CNS over time, we performed serial FMT – an optical imaging modality that allows for in vivo imaging of fluorescent probes through layers of skin, bone and tissue – of mice injected with recombinant monomeric human tau labeled with cypate, a near-infrared dye that can be easily conjugated to a number of different tracers, probes and molecules for optical imaging of tumors and other physiological processes [49‐52]. In this experiment, 4-6mo old WT and K14-VEGFR3-Ig mice were imaged at 1, 2, 24, 48, 72 and 168 h post-injection of tau (Fig. 2a). Fluorescence was quantified in the brain to calculate amount of tau retained at each time point. Signal for each mouse at each time point was normalized to the 1 h time point. As we imaged mice over several days, we observed a clear result: tau clearance is delayed from the brain in K14-VEGFR3-Ig mice. Significantly more tau is retained at 48 h (85% vs 52%) and 72 h (52% vs 22%) post-injection in the K14-VEGFR3-Ig mice, strongly suggesting that in the absence of dural lymphatics, extracellular tau is not as effectively cleared from the CNS to the periphery (Fig. 2b). This is reflected in the half-life of tau in both groups (Fig. 2d): tau has a shorter half-life of 39.67 h in WT mice compared to 154 h in the K14-VEGFR3-Ig mice.

To get a more clear understanding of tau clearance by lymphatics we repeated our experiment by injecting HSA-cypate to serve as a reference tracer. Since it is known that albumin is cleared by lymphatic vessels out of the CNS [31, 32], we hypothesized delayed clearance of HSA in K14-VEGFR3-Ig mice. Longitudinal FMT of 4–6 mo WT and K14-VEGFR3-Ig mice injected with HSA-cypate reveals that significantly more HSA is retained in the brain of the transgenic mice at 24 h (57% vs 29%) and 48 h (36% vs 15%) compared to WT mice (Fig. 2c), suggesting that similar to tau, clearance of HSA is delayed in mice lacking a functional dural lymphatic system. Additionally we found that half-life of HSA was longer in K14-VEGFR3-Ig mice (26 h) compared to WT mice (~ 13 h). This is in agreement with published reports of rat albumin’s half-life in rat brain [53, 54] being in the range of 11–18 h, thus further validating this method to measuring CNS clearance of proteins. The fact that HSA is cleared faster than tau could perhaps indicate uptake and/or additional extracellular retention of tau within CNS.

As we showed there is delayed clearance of extracellular tau from the brain, one would predict that the amount of tau appearing in the blood would also be delayed. In order to study this directly, we measured plasma tau in both groups of mice to complement FMT experiments looking at brain retention of tau to get a more complete picture of tau clearance. We used an antibody-mediated approach [44] to increase half-life of soluble tau in the plasma to allow us to measure it reliably. This was done because the half-life of tau once it reaches the plasma is only 8.49 min; however, in the presence of the anti-tau antibody HJ8.5, the half-life of plasma tau increases to 3.4 h allowing one to measure it there in a more facile fashion [44]. Following intraperitoneal antibody administration of HJ 8.5 (an anti-human tau antibody), WT and K14-VEGFR3-Ig mice were injected in the hippocampus with 1.1 μg tau. Blood was collected at 2, 24, 48, 72 and 168 h post-injection and plasma human tau was measured by a high-sensitivity ELISA on a Simoa analyzer. The plasma tau assay uses capture antibody HJ8.7 (which binds to N-terminal epitopes 117–122 of tau) and biotinylated detection antibody BT2 (which binds to epitopes 194–198 of tau). We found that K14-VEGFR3-Ig mice, which retained a significantly higher amount of tau in the brain, correspondingly also show delayed appearance of tau in the periphery (Fig. 3) compared to WT mice. The amount of human tau in the plasma peaked earlier in the WT mice than in the K14-VEGFR3-Ig mice (24 h vs. 48 h). Significantly higher plasma tau was present in K14-VEGFR3-Ig mice at 48 h, when the WT mice had already cleared most of the tau from the periphery. Together with the fluorescence data from the brain, these plasma tau measurements strongly suggest that impaired dural lymphatic function leads to increased retention in the brain as well as delayed clearance of tau to the periphery.