Abstract
Tauopathies are a hallmark of many neurodegenerative diseases, including Alzheimer’s disease and traumatic brain injuries. It has been demonstrated that amyloid-beta peptides, alpha-synuclein, and prion proteins cross the blood-brain barrier (BBB), contributing to their abilities to induce disease. Very little is known about whether tau proteins can cross the BBB. Here we systematically characterized several key forms of tau proteins to cross the BBB, including Tau-441 (2N4R), Tau-410 (2N3R), truncated tau 151–391 (0N4R), and truncated tau 121–227. All of these tau proteins crossed the BBB readily and bidirectonally; however, only Tau-410 had a saturable component to its influx. The tau proteins also entered the blood after their injection into the brain, with Tau 121–227 having the slowest exit from brain. The tau proteins varied in regards to their enzymatic stability in brain and blood and in their peripheral pharmacokinetics. These results show that blood-borne tau proteins could contribute to brain tauopathies. The result also suggest that the CNS can contribute to blood levels of tau, raising the possibility that, as suggested for other misfolded proteins, blood levels of tau proteins could be used as a biomarker of CNS disease.
INTRODUCTION
Tauopathies occur in approximately 20 different neurodegenerative diseases, including Alzheimer’s disease, progressive supranuclear palsy, and traumatic brain injuries [1]. The hallmark of a tauopathy is the abnormal deposition of tau proteins, usually in hyperphosphorylated and truncated forms, in brain tissues. Tau proteins belong to the family of intrinsically disordered proteins (IDPs). Under physiological conditions, tau as a cytosolic protein regulates axonal transport and is involved in microtubule assembly and stabilization [2]. Tau proteins also have synaptic functions and play important roles in DNA stabilization [3, 4]. Under physiological conditions these proteins lack 3-D structure and exist as dynamic ensembles of interconverting structure [5]. When stressed or upon interaction with other proteins, nucleic acids, cell membranes, or small molecules, IDPs fold either partially or completely. Without a binding partner, post-translational modifications such as truncation, hyperphosporylation, glycation, glycosylation, nitration, or deamidation promote IDPs to become misfolded, losing their conformational characteristics and normal functions. The best-characterized post-translational modifications of tau are hyperphosphorylation, truncation, or a combination of both, contributing to the transition of tau from a disordered highly soluble protein to an insoluble aggregate form [2, 6–8]. Protein aggregation, formation of fibrils, and toxicity are consequences of these modifications [9]. As such, tau proteins are included in a group of proteins whose misfolding is associated with disease states, including amyloid-beta (Aβ), α-synuclein, and prion proteins associated with Alzheimer’s disease, Parkinson’s disease, and the prion diseases, respectively [10].
The blood-brain barrier (BBB) is intimately associated with those other proteins and the diseases with which they are associated. Specifically, Aβ proteins cross the BBB in both the brain-to-blood and blood-to-brain directions [11, 12]. Two transport proteins, low-density lipoprotein receptor-related protein-1 and p-glycoprotein, transport the Aβ proteins in the brain-to-blood direction [13, 14]. These transporters are altered by oxidative stress and inflammation [15–17] and their impaired function is thought to promote Alzheimer’s disease by causing increased brain retention of the Aβ proteins [18, 19]. Conversely, Aβ can damage the BBB, leading to its dysfunction [20]; a similar role has been proposed for tau proteins [21, 22]. Prions are also capable of crossing the BBB from the blood [23]. Likewise, α-synuclein has also been shown to cross the BBB in both the brain-to-blood and blood-to-brain directions [24, 25]. A more recent study has demonstrated that blood-borne α-synuclein transported out of brain in exosomes is increased in Parkinson’s disease [26].
In contrast, little is known about whether tau proteins cross the BBB or otherwise interact with it, except that, in a very recent pilot investigation, we have demonstrated that one particular form of tau [27] can exit from the brain to the blood. To more further investigate the characteristics of tau transportation, here we examined four tau proteins for their ability to enter or exit the brain across the BBB; these four proteins are Tau-441 (2N4R), Tau-410 (2N3R), truncated tau 151–391 (0N4R), and truncated tau 121–227 (Tau 121–227).
MATERIALS AND METHODS
Tau proteins
Recombinant human tau proteins (Table 1) were purified on ÄKTA Protein Purification Systems (GE Healthcare Life Sciences, UK) from E. coli bacterial lysates according to the previously published method [28]. Briefly, the bacterial lysates were loaded onto HiTrap SP HP ion-exchange column. The fractions containing tau were pooled and separated by size exclusion chromatography on HiLoad 26/60 Superdex 200 preparative grade column (GE Healthcare, Seattle, WA). After the buffer exchange, the proteins were stored under argon atmosphere at –80°C [29].
Animal use
CD-1 male (8-week-old) mice (Charles River, Wilmington, MA) were given ad libitum access to food and water and were kept on a 12/12 h light dark cycle. All animal studies were performed at a facility that is approved by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC) and under protocols approved by the Institutional Animal Care and Use Committee (IACUC) of the Veterans Affairs Puget Sound Health Care System, Seattle, WA, USA.
Radioactive labeling
Tau-441, Tau 121–227, Tau 151–391, and Tau-410 were radioactively labeled with Na 125I (Perkin Elmer, Waltham, MA) by the chloramine-T (Sigma–Aldrich, St. Louis, MO) method. Albumin (Sigma–Aldrich) was radioactively labeled with Na131I (Perkin Elmer) by the chloramine-T method or with Tc99m (GE Healthcare) using stannous tartrate method (MP Biomedicals LLC, Solon, OH). Radioactively labeled Tau-441 (I-Tau-441), radioactively labeled Tau 121–227 (I-Tau 121–227), radioactively labeled Tau 151–391(I-Tau 151–391), radioactively labeled Tau-410 (I-Tau-410), and radioactively labeled albumin (I-Alb or Tc99m-Alb) were purified on a column of Sephadex G-10 (Sigma–Aldrich). Specific activities for the tau proteins ranged from about 50–150 mCi/g.
Blood-to-brain influx
Multiple-time regression analysis was used to measure the blood-to-brain rate of uptake
[30, 31]. Mice were anesthetized with an intraperitoneal (ip) injection of 0.15 mL of
urethane (40%) and a 200 μl injection of lactated Ringer’s solution (LR) containing
3×105 cpm of a radioactively labeled tau was injected into the jugular vein.
In some studies, 3×105 cpm I-Alb or Tc99m-Alb were co-injected to quantify
vascular space. Arterial blood was collected from the carotid artery after a time ranging
from 1 to 30 min and the brain was removed and weighed. The brain/serum ratios were
plotted against exposure time (Expt) using Prism 6.0 (GraphPad Inc, San Diego, CA), where
Expt was calculated with the formula:
Brain-to-blood efflux
A method based on intracerebroventricular (icv) injection was used to assess brain-to-blood efflux rate [32]. This method differs from another popular method, the brain efflux index [33], in that the icv method inventories both brain tissue and the choroid plexus and measures actual efflux rate rather than an index [34, 35]. Mice were anesthetized with an ip injection of 0.15 mL of 40% urethane (Sigma–Aldrich, St. Louis, MO). The scalp was removed and a hole was made into the skull (0.5 mm posterior and 1 mm lateral to the bregma) and 1 μl of LR containing 2.5×104 cpm of a radioactive tau was injected into the lateral ventricle of the brain using a 1.0 μl Hamilton syringe. In some studies, 2.5×104 cpm of I-Alb or Tc99m-Alb was co-injected as a control.
For efflux kinetics studies, mice were decapitated 2, 5, 10, 20, or 30 min after icv injection. For self-inhibition studies, 2.5×104 cpm of a radioactive tau with or without 1 μg of its unlabeled version was given by icv injection and mice decapitated 10 min later, except for Tau-410 for which, because of its faster efflux rate, mice were decapitated 5 min after icv injection. Cross-inhibition of I-Tau-410 transport by Tau-441 was also tested. The whole brain was removed and weighed. The level of radioactive tau available for transport at t = 0 was estimated by repeating this procedure in mice that had been overdosed with urethane and had been dead for 10–20 min. The levels of radioactivity in the brain samples were counted for 3 min in a gamma counter. The results were expressed as the percent of the icv dose that remained in a g of brain and the log of the mean (n = 3/time point) was plotted against time with the time curve being determined in duplicate.
Characterization of radioactivity extracted from brain and blood in mice
Radioactivity recovered from brain tissue and serum after the icv injection of a radioactive tau protein was characterized by TCA precipitation. Whole brains and serum were harvested on ice at 10-min post-injection, except for Tau-410 which because of its more rapid efflux rate was harvested at 5 min. Processing controls were generated by spiking radioactivity into collection tubes and harvesting organs from mice not receiving a radioactive injection. Brains were homogenized in 1 ml of ice-cold LR containing 1% albumin using a bead beater (Biospec) for 30 s at 5400 g. The tube was rinsed with another 3 ml LR containing 1% albumin. The homogenate was centrifuged at 10,000 g for 20 min, and the supernatant was collected. For acid precipitation of brain tissue, equal volumes (1.5 ml) of supernatant from the homogenate and 30% TCA were mixed at room temperature. For acid precipitation of serum, 50 μl of serum was mixed with 500 μl of 1% albumin/LR, and proteins were precipitated by adding 500 μl of 30% TCA. Precipitates were centrifuged at 5400 g for 10 min, and the pellets and supernatants were counted in a gamma counter. The percentage of radioactivity in the pellet reflects intact protein.
RESULTS
Blood-to-brain Influx
Figure 1 shows the results for blood-to-brain influx of the four tau species. All tested tau proteins showed uptake by brain with the most rapid entry being for Tau 151–391, which had a unidirectional influx rate, or Ki, of 0.448±0.005 μl/g-min. Tau 121–227 had the slowest uptake, which did not show a statistically significant relation between brain/serum ratios and Expt until corrected for the albumin space, a maneuver which greatly reduced the statistical variance. After correction for albumin space (typically 8–12 μl/g), the Ki was 0.113±0.020 μl/g-min. The influx rates for Tau-441 (0.145±0.054 μl/g-min) and for Tau-410 (0.209±0043 μl/g-min) were intermediate between those for Tau 151–391 and Tau 121–227.
Inhibition of transport was not saturable for any tau except for Tau-410 (Fig. 2). A 1 μg/mouse dose of Tau-441 did not affect the blood-to-brain transport of either Tau-410 (Fig. 2) nor of Tau-441 (not shown). Radioactivity extracted from blood and brain 10 min after the iv injection of I-Tau-410 was 100% and 96% acid precipitable, respectively.
Clearance rates from blood for Tau-441, Tau 151–391, and Tau 121–227 showed biphasic kinetics (Fig. 3). Clearance of Tau 151–391 was most robust among these three. Clearance of Tau-410 followed first order kinetics with a half time clearance of 22.8 min.
Brain-to-blood efflux
All tau proteins showed evidence of brain-to-blood efflux (Fig. 4). Tau-441 and Tau-410 efflux followed one phase decay kinetics. Tau 151–391 showed linear kinetics with a clearance rate of 86 min. Efflux of Tau 121–227 was so slow that the relation between log(% Inj/g-brain) and time was not significant. Such a slow efflux is suggestive of retention by braintissues.
Brain-to-blood efflux was further evidenced by the appearance of radioactively labeled tau proteins in blood after their icv administration (Fig. 5). Tau-410 showed the highest levels in blood with Tau 151–391 showing the lowest. These two tau proteins were also the least stable in blood after their icv injection (Table 2) and so their findings need to be interpreted with more caution.
No radioactive tau protein showed evidence of saturable efflux. However, radioactive Tau 121–227 did show increased efflux when co-injected with unlabeled Tau 121–227 as evidence by increased brain-to-blood transport (Fig. 6, upper panel) and by increased appearance in blood (Fig. 6, lower panel).
DISCUSSION
This work is the first to extensively characterize the ability of several tau species to cross the BBB. We chose four tau proteins to represent a variety of characteristics among those thought to be found in brain during health and disease (Table 1). Tau-441 is a full-length tau protein containing 4 repeats and is the longest known physiological form of tau. Tau-410 is an isoform containing only 3 repeats and is also considered physiologic. Tau 151–391 is a double truncated tau and both truncation points are present in human Alzheimer’s disease brain [36, 37]. Tau 121–227 is a proline-rich region of tau and is found in the CSF [38].
We found tau proteins cross the BBB in both the brain-to-blood and blood-to-brain directions. For Tau-410, we found a saturable component for the transport into brain. We found most tau proteins to be stable in blood and brain and that the tau proteins, especially Tau 121–227, are sequestered by brain. This bidirectional penetration of the BBB by the tau proteins suggests that blood-borne tau could contribute to brain levels of tau and that the CNS could be a source of peripheral tau. Below, we discuss each of these major findings.
All the tau proteins examined entered the CNS from the blood with the rate of transport varying about 3-fold among them. Substances cross the BBB by mechanisms that can result in such variations in uptake rate, including transcellular diffusion, adsorptive transcytosis, and the presence of a saturable transporter [39, 40]. We did find that Tau-410 has a saturable component to its transport, but the other tau proteins did not, nor was Tau-441 able to cross-inhibit the transport of I-Tau-410. The exact mechanisms used by the tau proteins to cross the BBB cannot be exactly determined from our results. However, transcellular diffusion is not likely as these tau proteins are substantially larger than CINC1, the largest molecule so far shown to cross the BBB by transmembrane diffusion [41]. Adsorptive transcytosis is a likely mechanism for the non-saturable tau proteins as it is often difficult to demonstrate saturability for substances crossing by this mechanism.
Differences in the rate of clearance from blood and enzymatic stability also varied among the tau proteins, but these factors tend to affect brain accumulation more than the influx rate [42]. Cerebral blood flow is unlikely to affect the brain uptake of these tau proteins as their influx rates were too low to make them flow dependent. These findings show that blood-borne tau could contribute to brain tauopathy.
Brain-to-blood transport, or efflux, is important as it can greatly influence brain uptake and retention of a substance [43]. It also provides a mechanism by which the brain can contribute to blood levels and so could explain why blood levels of tau are elevated in those with traumatic brain injury [44]. Efflux is also a major mechanism by which the brain can rid itself of a substance. For example, efflux of amyloid beta peptides from brain by LRP-1 and P-glycoprotein is a major mechanism by which they are cleared from the CNS [45–47]. Impaired clearance leads to increased brain levels of Aβ peptide and cognitive impairment [19] and so could contribute to the onset or progression of Alzheimer’s disease [18]. Here, we found that efflux occurred for all four of the tau proteins, although the rate of efflux was slow. The slowest mechanism for brain efflux is that of bulk flow (CSF reabsorption) and so this is likely an important mechanism for the tau proteins. However, for Tau 121–227, the rate was so slow as to be consistent with sequestration by brain. As demonstrated by both a decrease in brain retention and an increase in appearance in blood, the efflux rate of I-Tau 121–227 increased when unlabeled Tau 121–227 was included in the icv injection, consistent with a saturable sequestration of Tau 121–227 by brain. The tendency toward nonlinear efflux curves for the other tau proteins suggests that bulk flow is not the only mechanism influencing clearance from brain and sequestration could be an additional mechanism. These results suggest both that tau proteins can be retained by brain tissue and also that the CNS can contribute to blood levels of tau. This suggests that tau blood levels might be useful as a biomarker in a way analogous to that recently suggested for α-synuclein [26].
In this study, efflux was demonstrated for the tau proteins after their icv injections by both a disappearance over time from brain and appearance in blood. The use of this method is relevant, as a recent study reported that extracellular tau in the brain is largely cleared along the glymphatic paravascular pathways into the CSF and blood [48]. Our results imply that tau might be a substrate for a specific or saturable transporter system at the BBB. Given that the two mechanisms, glymphatic system and trans-endothelial efflux across the BBB via efflux transporters, occur within the same perivascular domains, it is plausible that these two processes interact with one another.
The differences among the tau proteins in their sequestration, influx, efflux, and degradation rates likely relates to their structural differences, although it is currently difficult to ascribe specifically how structure influences these parameters. The C-terminal domain of tau protein (assembly domain) binds to microtubules and promotes their assembly. Binding to microtubules occurs through repeated domains (R1-4R) encoded by exons 9–12. There are specific sequences that are strongly involved in the tau-microtubule interactions. These sequences include 240KSRLQTAPV248, 275VQINKKLDLS285, and 297IKHV300. The sequences 275VQINKKLDLS285 and 297IKHV300 are coded by exon 10 and this may explain why 4R tau isoforms interact with microtubules more strongly than 3R tau isoforms [49, 50] and could be related to the difference between Tau-441 (4R) and Tau-410 (3R) in their CNS influx and efflux observed in this study.
In summary, we found evidence that tau proteins can cross the BBB bidirectionally. This suggests both that blood levels could contribute to brain tauopathies and that the brain could contribute to the blood levels of tau.
