Research ArticleDISEASES AND DISORDERS

Experimental evidence for the age dependence of tau protein spread in the brain

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Science Advances  26 Jun 2019:
Vol. 5, no. 6, eaaw6404
DOI: 10.1126/sciadv.aaw6404

Abstract

The incidence of Alzheimer’s disease (AD), which is characterized by progressive cognitive decline that correlates with the spread of tau protein aggregation in the cortical mantle, is strongly age-related. It could be that age predisposes the brain for tau misfolding and supports the propagation of tau pathology. We tested this hypothesis using an experimental setup that allowed for exploration of age-related factors of tau spread and regional vulnerability. We virally expressed human tau locally in entorhinal cortex (EC) neurons of young or old mice and monitored the cell-to-cell tau protein spread by immunolabeling. Old animals showed more tau spreading in the hippocampus and adjacent cortical areas and accumulated more misfolded tau in EC neurons. No misfolding, at any age, was observed in the striatum, a brain region mostly unaffected by tangles. Age and brain region dependent tau spreading and misfolding likely contribute to the profound age-related risk for sporadic AD.

INTRODUCTION

Alzheimer’s disease (AD) is a progressive dementia typically starting with memory impairments and progressing to profound cognitive impairments. The incidence of AD is strongly age-related, with rare cases diagnosed before age 50 and with the number of cases doubling every 10 years until more than half of the population is affected by age 90 (1). The cognitive symptoms are largely attributed to the intraneuronal aggregation of tau (2) that appears first in entorhinal and hippocampal projection neurons and over time spreads to other limbic areas, neocortical association areas, and ultimately to primary sensory cortices (3, 4).

In the past years, the idea that tau protein can spread from one anatomical region to the next has been supported in experiments using transgenic mice, by viral constructs for a spatially restricted expression of mutant human tau in the brain (5, 6), or by direct injections of pathological tau protein isolated from transgenic mice or human AD brain into transgenic tau mice (711). We reasoned that, if age played a role for the propagation and aggregation of tau in the human brain, then this would also be reflected in animal models of tau propagation. We therefore developed a method to assess the effect of (i) the age of the animal, (ii) the specific brain region, or (iii) the aggregation/misfolding state of tau on the propagation of tau in the living mouse brain.

RESULTS

Adeno-associated viruses to visualize tau protein spread between neurons

We constructed adeno-associated viruses (AAVs) that allowed us to reliably distinguish between neurons that express human tau versus neurons that receive human tau through transmissive reception of human tau protein (huTau): The AAVs are coding one mRNA, green fluorescent protein (GFP)–2a-huTau (monocistronic), but produce an equimolar ratio of two individual proteins, GFP and huTau (Fig. 1A). The short 2a peptide sequence catalyzes a self-cleavage reaction that separates GFP and huTau during translation at the ribosome (12). As a result, neurons that are transduced with the virus (or received the mRNA via less common mechanisms such as episomal or exosome-associated RNA transfer) produce GFP and huTau as individual proteins. In contrast, neurons that receive huTau through cell-to-cell spreading of tau protein have huTau but no GFP. Such neurons can be identified in neuronal cultures or in brain sections by coimmunofluorescence labeling of huTau and GFP (fig. S1A). In our AAV constructs, we purposefully chose the location of GFP upstream of huTau because of the following reasons: In the case of dysfunctional 2a peptide cleavage, a cell would produce the fusion protein GFP-2a-huTau, which could, in principle, also travel between cells. However, the presence of GFP would classify donor and recipient cells of GFP-2a-huTau as GFP+ and therefore reject them from the group of tau recipient neurons. This diminishes the false positives in the system and increases the stringency of our analysis; we may, as a consequence, underestimate the amount of tau recipient neurons.

Fig. 1 Enhanced neuron-to-neuron propagation of mutant P301Ltau in vivo.

(A) Schematic showing the AAV sequence, the mRNA, and the proteins encoded in AAV CBA.eGFP.2a.huTau (WTtau and P301Ltau), as well as the tau protein propagation principle and detection methodology. Using a self-cleaving 2a peptide, transduced “donor” neurons express both eGFP and human tau as individual proteins. The propagation of tau can be visualized by immunofluorescence labeling of postmortem brain sections or fixed neurons in culture: Human tau detected in “recipient” neurons that do not express the fluorescence transduction marker eGFP indicates the propagation of tau between cells. Thereby, the upstream location of the GFP transduction marker prevents the detection of false positives that could occur due to incomplete translation of the mRNA. (B) Schematic for the unilateral injection of AAV eGFP-2a-huTau into the EC (green) and the experimental work flow. The location of the EC, the HPC (and subregion CA1), and the dentate gyrus (DG) in horizontal mouse brain sections is indicated. (C) Example of the immunofluorescence labeling of human tau (red) and GFP (green) in a horizontal brain section of a GFP-2a-WTtau–injected mouse with many huTau recipient cells: [huTau+/GFP] cells, which received tau by cell-to-cell propagation (white arrowheads in close-ups 1 and 2), can be seen adjacent to the EC injection side (1) and in synaptic connected area CA1 (2). Cells transduced with the AAV (GFP+) can, in this specific case, be found in the EC and CA3. (D) Quantification of tau propagation (no. of recipient cells/no. of transduced cells) was done by counting all recipient (huTau+, red) and donor neurons (GFP+, green) in the entire ipsilateral EC and HPC formation (dashed white line). A higher propagation (P = 0.0472) was detected for P301Ltau compared to WTtau. (E) Scatterplot shows the average of recipient versus donor cells per mouse. Mean ± SEM, n = 3 animals per group and n = 4 to 5 brain sections per animal; single data points represent the mean per animal; unpaired two-tailed Student’s t test with Welch’s correction.

First, we verified the function of the viral constructs in vitro [fig. S1, B and C; (13)]. As predicted, if we express tau in a small fraction of neurons, some nontransduced murine neurons become human tau positive by receiving tau protein through cell-to-cell transmission during the first 7 days of transduction (detected by immunostaining and flow cytometry of GFP+ and huTau+/GFP cells; fig. S1, B and C).

Tau protein spreads to adjacent and a few distal neurons in the mouse brain

The viral vectors were then used to explore the spreading of tau in neural circuits in vivo (Fig. 1, B to D, and fig. S2, A to D). Previous studies showed propagation of misfolded P301Ltau along a major neural pathway—from entorhinal cortex (EC) to dentate gyrus neurons—in transgenic mice after 15+ months (5, 6, 14). The propagation of pathological aggregated tau that is competent to seed tau aggregation in mutant tau transgenic mice occurs across connected brain regions as well (10, 11). We predicted that the spreading of tau proteins would respect anatomical pathways also when using our AAV approach and tested this idea by coinjecting AAV GFP-2a-P301Ltau with the established anterograde tracer, Phaseolus vulgaris leucoagglutinin (PHA-L), or the retrograde tracer cholera toxin B (CtB; fig. S3). As expected from previous reports, some recipient tau neurons appear in areas downstream of entorhinal neurons expressing GFP-2a-tau (PHA-L+) and in distal areas that are anatomically connected to the EC via long-distance projections; for example, the olfactory cortex and the contralateral EC and hippocampus (HPC) (fig. S4). However, many huTau recipient neurons were located adjacent to GFP-2a-tau donor neurons in the EC or in areas that seem to be retrogradely connected (CtB+; Fig. 1C and fig. S3). This indicates that AAV-driven expression of soluble human tau in EC neurons leads to some synaptic long-distance spread and to strong local spreading of tau protein to neurons in adjacent brain areas. Notably, local spreading of tau protein could be synaptic because adjacent neurons are connected by many local recurrent collaterals; we have not yet conclusively tested this.

Enhanced spreading of soluble nonaggregated mutant tau

One of the working hypotheses states that the propagation of tau in the brain is coupled to tau misfolding. Frontotemporal dementia (FTD)–associated point mutations in the MAPT gene, for example, P301L, P301S, or ΔK280, enhance the misfolding and aggregation propensity of tau (15) and are therefore used to introduce aggregation and neurofibrillary tangle (NFT) formation in transgenic tauopathy animal models (16). Here, we directly compared the spreading of wild type (WT) and P301Ltau after viral expression of GFP-2a-WTtau or GFP-2a-P301Ltau in the EC and some hippocampal neurons of adult WT mice (8 to 10 months of age). Reliable detection of individual tau recipient neurons, using their immunoreactivity for huTau but not for GFP, requires a very careful evaluation of the immunolabeled brain sections, which is extremely time consuming. For simplification and experimental feasibility, we decided to count tau recipient neurons only in the hippocampal formation and adjacent cortical areas (outlined in Fig. 1C). When comparing the spreading of tau (number of recipient neurons [huTau+/GFP] normalized to the number of transduced neurons [GFP+] to account for animal-to-animal differences in transduction efficiency), we found more spreading for P301Ltau compared to WTtau after 8 weeks (WTtau: 2.85 ± 0.28%, n = 3; P301Ltau: 4.04 ± 0.31%, n = 3; P = 0.0472; Student’s t test with Welch’s correction; Fig. 1D); the number of recipient neurons varied between animals (on average 17 to 121 recipient neurons per brain section per mouse) and correlated with the number of transduced neurons, and this observation seemed somewhat independent of the amount of transduced neurons.

Next, we hypothesized that GFP-2a-P301Ltau–expressing mice would have more misfolded or aggregated tau than WTtau-expressing mice and, at the same time, tested whether tau misfolding would be necessary for tau propagation. When analyzing the aggregation state of tau in the injected mice using the Alz-50 antibody that recognizes a conformational tau epitope present in AD, P301L-expressing donor neurons in the EC were consistently immunoreactive for misfolded tau [fig. S5A; (17)] and showed aggregation seeding potential in a cell-based tau aggregation assay [fig. S5B; (18)]. In contrast, WTtau-expressing neurons showed Alz-50 reactivity only after prolonged expression (i.e., 40 weeks in fig. S5A). We did not observe obvious neuronal death even after long-term expression of the GFP-2a-P301Ltau (fig. S5C). Notably, even 40 weeks after transduction, no β sheet containing tau aggregates (positive for the tangle dye Thiazine Red) could be found in P301Ltau-expressing neurons (fig. S5D), suggesting that misfolded tau accumulations in the donor cells can persist but do not rapidly and inevitably progress into amyloid-like β sheet structures or lead to neuronal death. Alz-50+ misfolded tau was not detected in recipient neurons in either P301Ltau- or WTtau-injected mice. These data suggest that misfolding of tau in the donor cells can enhance tau propagation, but tau misfolding/aggregation is not a prerequisite for propagation. These findings are further supported by work from the Buée laboratory showing the propagation of WT human tau after lentiviral expression in rat brain (19).

Enhanced P301Ltau spreading without changes in glial cell activation and proteostasis

In the brain, glial cells remove accumulating misfolded proteins including tau (20) and are part of the response to neurotoxic insults (21). Microglia may also be involved in tau propagation (22). Differences in microglia and astrocytes between WTtau- and P301Ltau-expressing mice could therefore explain the accumulation of misfolded tau and the elevated tau spreading in P301Ltau-expressing animals. By stereology, both WTtau- and P301Ltau-expressing mice had a similar increase of microglia (Iba1+) in the HPC adjacent to the tau-expressing EC (WTtau: 1.63 × 10−4 ± 0.2 × 10−4 cells/μm2, n = 4; P301Ltau: 1.71 × 10−4 ± 0.43 × 10−4 cells/μm2, n = 3; P = 0.7460; unpaired Student’s t test; fig. S6A). The amount of microglia was therefore not related to tau propagation in our model. By Western blot, increased astrocytic glial fibrillary acidic protein (GFAP) was detected in two of three P301Ltau-expressing ECs, although this failed to reach significance (fig. S6B). Together, we cannot rule out that differences in astrocyte activity contribute to the spread of tau in P301Ltau- versus WTtau-expressing mice, a phenomenon that has been reported elsewhere (23, 24).

The intracellular misfolding of proteins can be triggered by deficits in proteostasis mechanisms; the amount of misfolded proteins increases upon decrease in chaperone activity, upon reduced protein degradation via the proteasome (which is activated upon ER stress followed by the unfolded protein response), or upon reduced autophagy (25). We tested whether these mechanisms contributed to the observed increase in P301Ltau protein misfolding and spreading, but found no obvious changes in autophagy (Lamp3) and proteasome (PSMD13) markers in the injected EC of P301Ltau compared to WTau-expressing mice (fig. S6C). P301Ltau-expressing mice seemed to have less chaperone activity (Hsp70) and less phospho-tau (sS396/pS404 and pS262/pS356; fig. S6D) compared to WTtau in the EC. These findings were somewhat surprising since we suspected that tau misfolding, which is enhanced for P301Ltau, would be associated with increased chaperone activity to compensate for the increase in misfolded proteins. However, the lower levels of misfolded tau in WTtau-expressing neurons could be a consequence of the higher Hsp70 levels in these mice, similar to what has been observed in a previous study (26). Tau misfolding is also coupled to tau hyperphosphorylation in the human AD brain. However, it may be that higher levels of phospho-tau, at least at the tested AD-relevant epitopes pS396/pS404 and pS262/pS356, are in fact more relevant for the Hsp70 chaperone response than tau misfolding.

Both WT tau hyperphosphorylation and tau misfolding are indicators of tau toxicity in the brain, which can manifest in synapse loss (27, 28) or DNA damage (29). We did not find differences in synapse markers (PSD95 and synapsin 1) between WTtau-expressing ECs (higher phosphorylation) versus P301Ltau-expressing ECs (increased misfolding), but DNA damage (H2a.X) seemed to be increased in P301Ltau-expressing ECs (fig. S6, E and F). In summary, our findings suggest that the increased propagation of P301Ltau is likely not caused by higher intracellular tau levels because of decreased tau degradation or by changes in the total synaptic pool; instead, the action of misfolded tau on the genomic DNA may contribute to the observed increased propagation.

Tau protein spreads faster in the old brain

Next, we explored the dependency of tau propagation and misfolding on age. Given the stronger spreading efficiency of P301Ltau, we focused on this construct. In transgenic mice, detectable P301Ltau propagation occurs in old animals (~18+ months) (5, 6). It remains unclear though whether the molecular event of tau transfer between cells is too rare for an earlier detection, which therefore only becomes possible with extended time, or whether aged animals provide a neural system environment that is more permissive for tau propagation events. To answer this question, we injected young (3 months old) and old (22 to 24 months old) mice with AAV GFP-2a-P301Ltau in the EC and counted all individual huTau recipient neurons (huTau+/GFP neurons) and all donor neurons having GFP (huTau+/GFP+ neurons) in the entire hippocampal formation and adjacent cortical areas (Fig. 2A). Both young and old animals showed some recipient neurons after 12 weeks. Consistent with the hypothesis that the aged brain provides a milieu that is more permissive for tau spread than the young brain, the spreading rate (no. of recipient neurons/no. of transduced neurons) in old animals was about twofold higher than that in young animals (Fig. 2B; young: 0.41 ± 0.06%, n = 4; old: 1.01 ± 0.19%, n = 5; P = 0.0322; Student’s t test with Welch’s correction). The amount of transduced donor cells (GFP+) in the EC was variable but similar in young and old mice and correlated with the number of recipient neurons (Fig. 2, C and D, and fig. S7A; on average 2.5 to 12 recipient neurons per brain section per mouse).

Fig. 2 Propagation and misfolding of tau are enhanced with age.

(A) Representative images of human P301Ltau propagation after AAV GFP-2a-P301Ltau injection into the EC in young and old mice. For analysis, huTau recipient neurons (white arrowhead in CA1 close-up) were counted in the EC, the hippocampal formation, and the adjacent cortex (ROI outlined with dashed white line). (B) Tau propagation (no. of recipient cells/no. of transduced cells) in old animals was still small but threefold higher (P = 0.0322) compared to young adult animals (12 weeks after injection). (C) The amount of transduced GFP+ neurons in the EC of young and old mice relative to the amount of total neurons (DAPI+ neuronal nuclei) was determined by counting nuclei and GFP+ cells in the same area of EC in parallel. (D) Number of neurons (large DAPI+ nuclei; smaller glia nuclei were neglected) in the injected (ipsi) and noninjected (contra) EC of young and old mice reveals no difference in neuronal numbers. (E) Number of microglia (Iba1+) in the HPC of the ipsilateral and contralateral hemisphere shows no difference between young and old mice. (F) GFAP levels in EC/HPC extracts from young and old mice (6 weeks after injection) suggest more activated astrocytes in the ipsilateral side of both young and old mice, compared to the contralateral side. (G) Western blot analysis of EC/HPC extracts from noninjected young and old mice reveals no general difference in proteostasis, in the ER stress marker CHOP, or in autophagy markers LC3B and p62. (H) Images showing transduced neurons (GFP+, green) in the EC having misfolded tau (Alz-50+, pink) in an old and a young animal. (I) The amount of Alz-50+ neurons in the EC (normalized to area covered by transduced GFP+ neurons) appears to be ~2-fold higher in old animals (ns, not significant; P = 0.1531). Data presented as mean ± SEM. For cell counts, n = 4 young and 5 old animals and n = 3 to 5 brain sections per animal; single data points represent the mean per animal. For brain lysates, n = 5 young and old animals. Two-tailed Student’s t test with Welch’s correction when comparing two groups, and one-way ANOVA with Sidak’s correction when comparing multiple groups.

Neuronal death can lead to the bulk release of intracellular tau, which could boost the spreading of tau. However, young and old mice had similar numbers of neurons in the injected and noninjected EC (Fig. 2D), showing that no overt neuronal death occurred upon GFP-2a-P301Ltau expression in either group; we therefore exclude the possibility that the higher tau spreading in old mice resulted from neuronal death of transduced neurons in these animals. In fact, old mice had more recipient cells than young mice compared to the number of donor neurons (fig. S7A).

Differences in glial cell activation or deficits in proteostasis in old mice could lead to an accumulation of tau in the donor EC neurons and thereby promote the release and spreading of P301Ltau in older animals. We tested these ideas but did not find differences between young and old animals, in the amount of hippocampal microglia cells (Fig. 2E) and entorhinal astrocyte activation (GFAP in EC extracts; Fig. 2F), or in markers for autophagy (LC3B and p62) or ER stress (CCAAT/enhancer-binding protein-homologous protein, or CHOP, which is upstream of the unfolded protein response and proteasome activation; Fig. 2G and fig. S7B). A larger accumulation of tau in the donor neurons of old mice may, however, contribute to the enhanced spread, even in the absence of homeostasis changes. Other changes could also lead to an increase of tau spreading with age, for example, age-related changes in the extracellular matrix (30), which likely has a large influence on tau release and uptake; changes in unconventional protein secretion mediated by sulfated proteoglycans (31); or changes in gene transcription regulation (32, 33). We also tested the level of DNA damage (H2a.X) but did not find an increase in old P301Ltau-expressing mice (fig. S7, B and C); in the future, more detailed investigations of these possibilities are needed.

To examine whether tau misfolding would also be enhanced in the old brain (the second part of our hypothesis), we compared the number of EC neurons having misfolded tau (Fig. 2H) in young versus old mice. Some old mice have ~3 times more Alz-50 immunoreactive neurons in the EC, although this did not reach statistical significance [Fig. 2I; young: (2.65 ± 1.4) × 10−5 cells/μm2, n = 4; old: (4.97 ± 2.87) × 10−5 cells/μm2, n = 5; P = 0.1857; Student’s t test]. Considering our finding that old mice do not seem to have major deficits in protein homeostasis, it might be that some other unknown age-dependent factors are involved in the enhanced tau misfolding.

Misfolding of tau depends on the brain region

We were very intrigued to see that injections of AAV GFP-2a-P301Ltau into the striatal caudoputamen (CPu), a brain area that is not—or is very late—affected by NFT pathology in sporadic AD, did not yield P301Ltau misfolding in the same mice (Fig. 3), although both the EC and the CPu received the same amount of virus and showed similar levels of GFP-2a-P301Ltau expression in the injection sites (fig. S7D), as well as on a single-cell level (fig. S7E). Notably, in EC neurons, the level of GFP fluorescence (a proxy for P301Ltau expression level) did not correlate with tau misfolding (Fig. 3B; neurons with low GFP signal are positive for Alz-50), and neurons with similar GFP intensity were Alz-50+ in the EC but not the CPu. These data suggest that tau misfolding in EC neurons is not only an effect of intracellular P301Ltau level. One possibility to explain this observation is an inherent difference between the two neuronal populations that allow (or promote) tau misfolding in EC neurons, but not in CPu neurons. These differences could be encoded in the physiology, the metabolism, the architecture, or the gene expression of other cellular pathways of the neuronal populations in different brain structures. For example, the levels of tau kinases and phosphatases seem to be different in the two brain structures (34), which may contribute to differential phosphorylation and misfolding.

Fig. 3 P301Ltau misfolding in EC but not striatal neurons.

(A) Injection schematic and representative immunofluorescently labeled horizontal brain section of mice that were injected in both the EC and the striatum (CPu). Reference images of brain sections (coronal and horizontal) were taken from the MBL mouse atlas (www.mbl.org/atlas232/atlas232_frame.html). (B) Brain sections and close-ups of P301Ltau-expressing neurons in the injection sites in the EC and the striatum. Neurons with misfolded tau (Alz-50+) in the cell body can be found in the EC but not in the CPu, a region that, in most cases, does not develop tau pathology in AD. Notably, the amount of P301Ltau expression (approximated from GFP intensity) does not correlate with Alz-50 reactivity in the EC or CPu.

DISCUSSION

We introduced a newly developed AAV tool, AAV GFP-2a-tau, that allows the detection of tau (or other) protein spread in the living brain in versatile ways and more efficiently than previous techniques. Notably, this model differs conceptually from models that investigate the propagation of tau pathology (tau hyperphosphorylation, aggregation, and NFT formation) in the brain upon injection of pathological tau material; those models have previously made great strides in proving the seeded aggregation of tau in different connected brain areas. In contrast, the tool we present in this study enables us to determine the spreading of tau proteins independent of their misfolding state and shows that the previously reported propagation of seeded tau aggregation may underestimate the degree of soluble nonaggregated tau spread in the brain. In addition, the viral GFP-2a-tau vectors allow us to gain experimental traction on the effect of age and brain region on neurodegeneration-related tau phenotypes in the mammalian brain: spreading, misfolding, and brain region selectivity. By injecting AAV GFP-2a-huTau into differently aged WT animals, and thereby dissociating age from the duration of transgene exposure, we showed that age has a profound effect on tau protein spreading efficiency. Given that tau spreading per se is a rare event, even in neural systems overexpressing proaggregant forms of tau in transgenic or AAV-injected mice, the ~2-fold increased rate of spreading seen over just 12 weeks in aged mice could play a substantial role for the progression of tau pathology in older individuals.

Cell-to-cell protein transmission (in most cases) relies on the cellular release and uptake of proteins, and neuronal uptake of misfolded/aggregated tau relies on endocytosis, whereas uptake of soluble tau is additionally facilitated by macropinocytosis (35). Considering that we found most of the propagated tau (WT and P301L) to be soluble and not aggregated (neither Alz-50+ nor Thiazine Red+), this can explain the difference we observed in the tau spreading pattern (local soluble tau transmission) compared to studies that observed pathological tau aggregation/misfolding in downstream brain areas after injection or infusion of preaggregated tau into mice expressing aggregation-prone tau mutants (911); the propagation of soluble, nonaggregated tau has not been tested in these models. Our data indicate that the propagation of tau pathology in the brain not only could be facilitated by neuron-to-neuron transmission of misfolded seeding-competent tau but also may result from cell autonomous conditions (or cascades) that enable the misfolding of tau in certain brain areas. However, the number and the species of tau that travels across synapses remain unknown, mainly due to technical challenges.

The underlying reason for increased tau spreading in aging remains unclear. We did not find evidence of increased glial cell activation or deficits in proteostasis mechanisms, which could be expected to decrease during aging and thereby enlarge the pool of misfolded and aggregated proteins in the brain. Thus, there may be alternative drivers of tau propagation; we suspect that inherent cell autonomous conditions, which are specific for certain neuronal subpopulations in the brain, can predispose a cell for tau reception and misfolding. For example, changes in the gene transcription in individual cells, caused by DNA damage or altered heterochromatin organization, could play a role in this process. Activity dependence of tau propagation has recently been shown in mice (36); however, at this point, we cannot comment on whether a related mechanism underlies the difference in tau propagation we detect in WTtau- versus P301Ltau-expressing mice, and in young versus old mice. Notably, in a recent study, we showed that both WTtau- and P301Ltau-expressing transgenic mice have a similar reduced neuronal baseline activity (37).

It is important to note that P301Ltau-expressing EC neurons, despite having misfolded tau (Alz-50+), did not develop NFTs over a fairly extended time frame. Also important is the fact that tau recipient neurons did not develop Alz-50 reactivity. These findings suggest that the kinetics of tau spreading can be dissected from the kinetics of misfolding and aggregation at multiple points in the process: (i) Tau molecules can spread without being misfolded; (ii) tau misfolding or aggregation in donor neurons is not necessary for, but may enhance, tau spreading; and (iii) tau misfolding does not need to lead to NFT-like tau aggregation. These results challenge recent data, which suggest that even single molecules of misfolded tau are sufficient, in vitro, to initiate an irreversible cascade of tau misfolding and aggregation (38). It appears that under complex in vivo conditions, neurons seem to be capable of maintaining tau in a misfolded, nonaggregated state, which reinforces the possibility for a potential therapeutic window, in which one could stop the spread of tau pathology by targeting the propagation of tau regardless of its conformational state.

MATERIALS AND METHODS

Animals

All procedures were performed following the guidelines of the Institutional Animal Care and Use Committee and in compliance with the Animal Welfare Act, the Guide for the Care and Use of Laboratory Animals, the Office of Laboratory Animal Welfare, and the guidelines of Massachusetts General Hospital. The animals’ living conditions, including housing, feeding, and nonmedical care, were maintained by the house internal animal facility (CCM). For the comparison between GFP-2a-WTtau– and GFP-2a-P301Ltau–expressing mice, 8- to 10-month-old male WT (C57BL/6, the Jackson Laboratory) mice were used. For the comparison of young (3 months old) versus old (22 to 24 months old) mice, male C57BL/6 WT mice of the respective ages were purchased from the National Institute of Aging (NIA).

Primary neuron cultures

Primary embryonic mouse neuron cultures were prepared from freshly dissected embryonic cortices, as described previously. Briefly, pregnant female CD-1 mice (Charles River) were euthanized at embryonic days 14 to 16, embryonic cortices were dissected and homogenized using a papain dissociation kit (Worthington Biochemical), and neurons were plated on culture dishes that were pretreated with poly-l-lysine. For tau propagation studies, neurons were transduced by the direct addition of AAV enhanced GFP (eGFP)–2a-WTtau or eGFP-2a-P301Ltau particles to the culture medium at 7 days in vitro (DIV), and neurons were fixed for immunostaining at 14 DIV or detached with StemPro Accutase Cell Dissociation Reagent (Thermo Fisher Scientific) from the culture dish for flow cytometry at 14 or 21 DIV.

AAV design, cloning, and production

The cloning of eGFP-2a-WTtau and eGFP-2a-P301Ltau under the ubiquitous chicken β actin (CBA) promotor was performed as described previously (13). The plasmid DNA of both eGFP-2a-huTau constructs and GFP were then tested for inverted terminal repeat integrity by digestion with the restriction enzyme Sma I, DNA of sufficient quality was packaged into an AAV2/8 [titers ~0.6 × 1013 virus particles/ml; Massachusetts Eye and Ear Institute vector core], and active AAV stock was aliquoted and stored at −80°C to prevent freeze-thaw cycles.

Intracranial injections

AAVs encoding eGFP, eGFP-2a-WTtau, or eGFP-2a-P301Ltau were injected unilaterally into the EC and/or striatum (CPu) of WT mice (per injection site: 3.0 μl of AAV into the EC for WTtau and P301Ltau comparison, and 1.0 μl of AAV for age and brain region comparison; three to five mice per group). Injections were performed as described previously under standard aseptic surgery conditions: Animals were anesthetized with isoflurane (3% induction and 2% maintenance), a midline incision of the skin was made above the injection sites, and burr holes were drilled through the skull at the selected coordinates [coordinates (from bregma) for EC injections: anterior/posterior (A/P): −4.7 mm, medial/lateral (M/L): ±3.3 mm, dorsal/ventral (D/V): −2.0 mm from brain surface; coordinates for striatum (CPu) injections: A/P: +0.2 mm, M/L: ±2.0 mm, D/V: −2.6 mm from brain surface]. After lowering the needle into the brain to the injection location, AAV solutions were injected at a flow rate of 0.2 μl/min. In the case of coinjections of AAV with neural tracers PHA-L (rhodamine-labeled PHA-L, Vector Laboratories) and CtB (Sigma-Aldrich), 1.0 μl of AAV solution was mixed with 1.0 μl of each tracer solution, and 3 μl of the mix was immediately injected as described. A 10-μl Hamilton syringe with a 30-gauge beveled needle that was coupled to an injector pump was used. The injector was attached to a stereotaxic frame, in which the mice were head-fixated. After finishing the injection, the needle was left in place for 2 min to allow the diffusion of the injected AAV solution. Afterward, the skin over the injection site was sutured, and the animals were allowed to recover from anesthesia on a 37°C warming pad before returning them into a clean home cage. For analgesia, all mice received a subcutaneous injection of buprenorphine (0.05 mg/kg) immediately after AAV injection and were treated with Tylenol (in drinking water) for 3 days after the surgery.

Brain tissue and lysates

For lysates of EC and HPC, mice were perfused with phosphate-buffered saline (PBS), and then we extracted the brain, dissected the EC and posterior HPC of the injected and noninjected hemisphere on ice, and snap-frozen the tissue in liquid nitrogen and stored it at −80°C. Tris-buffered saline (TBS) extracts were prepared by homogenizing the tissue in three times (v/w) TBS containing protease inhibitor (cOmplete Mini, Roche) manually with a handheld Eppendorf tube homogenizer (30 to 40 strokes on ice), followed by centrifugation at 10,000g for 10 min at 4°C, and the supernatant was taken as the cytosolic brain extract; this fraction was used for the human embryonic kidney (HEK) cell seeding assays and for Western blots. The total protein content of nuclear enriched fraction and of brain extracts was determined using a bicinchoninic acid assay (Pierce).

Immunofluorescence labeling

For immunofluorescence labeling of brain sections, injected mice were perfused with PBS containing 4% paraformaldehyde (PFA). The whole brains were extracted and postfixed in 4% PFA/PBS for 3 days at 4°C and then cryoprotected in 30% (w/v) sucrose in PBS for 3 days, cut horizontally into 40-μm-thick brain sections on a freezing microtome, and stored in PBS/50% glycerol at −20°C. For immunostaining, the floating brain sections were washed briefly in PBS and then permeabilized with 0.2% Triton X-100/TBS for 20 min at room temperature, blocked in 5% normal goat serum (NGS)/PBS for 1 hour at room temperature, and then incubated with primary antibodies diluted in 3% NGS/PBS overnight at 4°C: chicken anti-GFP (1:1000, Aves), mouse anti–human tau Tau13 (1:1000, BioLegend), rabbit anti–human tau TauY9 (1:1000, Enzo Life Sciences), mouse immunoglobulin M (IgM) anti-misfolded tau (1:500, Alz-50, provided by P. Davis), and goat anti-CtB (1:1000, Millipore). After washing three times with PBS, secondary antibodies were diluted in 3% NGS/PBS and applied for 1.5 hours at room temperature: Alexa 488 anti-chicken, Cy3 anti-mouse, Cy3 anti-rabbit, Alexa 647 anti-mouse IgM, and Alexa 647 anti-goat (1:1000, Thermo Fisher Scientific). After three washes in PBS, sections were mounted on microscope glass slides with mounting media containing 4′,6-diamidino-2-phenylindole (DAPI) (Southern Biotech).

Thiazine Red staining of tangles was done by applying 0.05% (w/v) Thiazine Red (Sigma-Aldrich) dissolved in PBS (GIBCO) to permeabilized brain sections for 20 min at room temperature, followed by extensive washing in PBS (four times for 10 min and once overnight) to remove unspecifically bound dye. Imaging of immunolabeled sections was done using 5×, 10×, or 20× objectives on a Zeiss Axiovert equipped with a QuickSnap camera or on an Olympus BX51.

For flow cytometry experiments, directly labeled Tau13–Alexa 647 was produced by incubating anti-human tau antibody (100 μg) Tau13 (BioLegend) with active N-hydroxysuccinimide–Alexa 647 (solved in dimethyl sulfoxide, Thermo Fisher Scientific) for 1 hour at room temperature in PBS (pH 7.5). Excess dye was removed by dialysis of the antibody/dye mix in dialysis cassettes (Slide-A-Lyzer, molecular weight cutoff, 3000; PIERCE) against 2 liters of PBS overnight at 4°C.

Western blot analysis of brain lysates

For Western blot analysis of various proteins in EC/HPC extracts, 10 to 20 μg of total protein per lane was loaded on 4 to 12% bis-tris SDS–polyacrylamide gel electrophoresis (SDS-PAGE) gels (Invitrogen) and separated by SDS-PAGE using MES or Mops running buffers (Invitrogen). After blotting on nitrocellulose membrane (Amersham), the membranes were blocked in PBS-based blocking buffer (LICOR) for 1 hour at room temperature and then incubated in primary antibody solutions overnight at 4°C. Antibodies in blocking buffer were as follows: mouse anti-human tau Tau13 (1:1000, BioLegend), chicken anti-GFP (1:1000, Aves), rabbit-anti GFAP (1:1000, Abcam ab7260), rat-anti Hsc70 (1:1000, Abcam ab19136), rabbit-anti Hsp70 (1:1000, Abcam ab79852), mouse-anti CHOP (alternative name DDTR1, 1:1000, Abcam ab11419), rabbit-anti PSMD13 (1:1000, Abcam ab229812), rabbit-anti Lamp3 (1:1000, ab83659), rabbit-anti LC3B (1:1000, Abcam ab51520), rabbit-anti p62 (1:1000, Abcam ab109012), mouse anti–phospho-tau PHF1 (1:1000, provided by P. Davis), mouse anti–phospho-tau 12e8 (1:1000, Prothena), goat anti-PSD95 (1:1000, Abcam ab12093), mouse anti–synapsin 1 (1:1000, Millipore MABN894), H2a.X (1:500, Abcam), and mouse anti-actin (1:1000, Millipore MAB1501R). After three washes in 0.02% Tween 20/TBS, the membranes were incubated with secondary antibodies in blocking buffer: goat anti-mouse/rabbit/rat-680 and donkey anti-mouse/rabbit/chicken/goat-800 (each 1:3000, LICOR, Rockland) for 2 hours at room temperature. Protein bands were visualized using a LICOR infrared scanner at wavelengths of 680 and 800 nm.

Stereological cell counts

The number of huTau donor cells, huTau recipient cells, Alz-50+ neurons (misfolding), and DAPI+ nuclei (to assess cell loss) in the EC, subiculum, and hippocampal formation of AAV-injected hemispheres was determined by counting all [GFP+], [huTau+/GFP], and [Alz-50+] cells in the area of interest, which was outlined as region of interest (ROI) using the CAST software (Olympus). The cell numbers were counted in three to four brain sections per mouse and three to five mice per group. For DAPI+ nuclei counting in the EC, we outlined layers 1, 2/3, and 4/5 of the EC and performed stereological counting of 20% of the nuclei in the ROI, followed by extrapolation to 100%. Counting was done on an Olympus BX51 light microscope equipped with a 20× objective.

Analysis of endogenous GFP fluorescence

Fixed brain sections were incubated with DAPI for 15 min and mounted on glass slides (without immunostaining), and images of whole brain sections were taken using a slide scanner with a 20× objective (VS120, Olympus). For analysis, areas with GFP fluorescence in EC and CPu were outlined in each brain section and the mean ± SD fluorescence intensity of the areas was measured using ImageJ. For single-cell fluorescence, individual cell bodies were outlined manually in the EC and CPu injection sites, and their fluorescence intensity was measured.

Flow cytometry of huTau recipient neurons

To gently detach primary neurons from culture dishes (six-well plates, Corning), each well was rinsed with 2 ml of sterile warm D-PBS+/+ (GIBCO) and then incubated in 1 ml of prewarmed accutase (StemPro Accutase Cell Dissociation Reagent, Thermo Fisher Scientific) in the incubator for 1 hour. Using a 1-ml tip, the cell suspension was homogenized, transferred into a sterile 15-ml conical tube on ice, and centrifuged at 300g for 10 min at 4°C, and the cell pellet was resuspended in 2 ml of D-PBS+/+ containing 0.5% BSA. After pelleting of the cells as before by centrifugation at 300g, cells were resuspended in 1 ml of D-PBS+/+ with 0.5% BSA, and 2% PF/PBS (1 ml) was added for 10 min at room temperature to fix the cells. After washing the cells with PBS/0.5% BSA, cells were resuspended for permeabilization in 0.1% saponin (0.5 ml) in DPBS+/+ with 0.5% BSA, and 1 μl of direct-labeled Tau13-Alexa 647 antibody was added for 30 min in the dark at 4°C. To remove excess antibody, cells were washed once in 2 ml of DPBS+/+ with 0.5% BSA and finally resuspended in 1 ml of DPBS+/+. Flow cytometry of huTau recipient neurons was performed on a MACSQuant VYB flow cytometer (Miltenyi Biotec). The gating for neurons having GFP and/or huTau-Alexa 647 was done using neuronal cultures transduced with AAV GFP for [GFP+ and huTau] cells and with nontransduced neurons for background fluorescence. Cells were gated first on the basis of forward- and side-scatter to exclude debris and select for singlets. The proportion of Tau13+ cells with and without GFP was then compared.

HEK cell tau aggregation assay

HEK293 cells that stably express CFP/YFP-TauRD-P301S (TauRD, repeat domain amino acids 244–372 of 2N4R human tau; P301S, FTD mutation) were maintained under usual cell culture conditions in Opti-MEM (Gibco) supplemented with 5% FBS. For the experiments, cells were plated into eight-well glass-bottom dishes or 96-well plates at 50 to 60% cell density. Then the number of cells with aggregates was counted after treatment with [EC/HPC] extracts from mice injected into the EC. Tau seeding activity of TBS brain extracts was tested by applying 2.0 μg of total protein per well in a total of 200 μl of Opti-MEM containing 1% Lipofectamine 2000 (Invitrogen) in eight-well dishes and in a total of 50 μl of Opti-MEM with Lipofectamine in 96-well plates. After 30 hours, cells were washed with PBS, fixed with 4% PFA/PBS for 10 min at room temperature, and then imaged using a 20× objective on an Axiovert microscope.

Statistical analysis

To compare cell numbers and propagation rates, we determined the average cell number or cell percentage (no. of recipients/no. of donors) per mouse from three to four brain sections in three to five mice per group. For Western blots, three to five mice per group were analyzed for injected animals. Statistical analysis of differences between groups was performed using GraphPad Prism 6; groups were compared using unpaired two-tailed Student’s t tests with Welch’s correction, and confidence intervals of 95% were used. Comparison of more than two groups was done using one-way analysis of variance (ANOVA) with Sidak’s or Tukey’s test for multiple comparison. All values are given as mean ± SEM. Raw data are available online after publication of the manuscript.

SUPPLEMENTARY MATERIALS

Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/6/eaaw6404/DC1

Fig. S1. Detection of tau protein propagation using AAV GFP-2a-huTau.

Fig. S2. Unilateral expression of AAV GFP-2a-huTau in EC and HPC of the mouse brain.

Fig. S3. Labeling of areas connected to the AAV expression site in the EC.

Fig. S4. Tau propagates to distally connected areas after GFP-2a-WTtau expression in the EC.

Fig. S5. Enhanced misfolding in the absence of aggregation in P301Ltau-expressing EC neurons.

Fig. S6. Proteostasis, phospho-tau, and neurotoxicity markers in WTtau- and P301Ltau-expressing ECs.

Fig. S7. P301Ltau propagation, Western blots, and comparison of GFP-2a-P301Ltau expression in young and old mice.

This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license, which permits use, distribution, and reproduction in any medium, so long as the resultant use is not for commercial advantage and provided the original work is properly cited.

REFERENCES AND NOTES

Acknowledgments: Funding: S.W., R.E.B., and B.T.H. received funding from the Ruth K. Broad Biomedical Research Foundation, BrightFocus Foundation, MGH, and the NIA. S.W. received additional funding from Deutsches Zentrum für Neurodegenerative Erkrankungen (DZNE) of the Helmholtz Foundation. B.T.H. received additional funding from the Tau Consortium and the Coins For Alzheimer’s Research Trust (CART). Author contributions: S.W., R.E.B., and B.T.H. designed study concept and experiments. S.W. and R.E.B. performed most of the experiments. L.D., A.B.R., A.C.A., M.H., D.M., M.J.K., J.S., and N.T. helped with experiments. Z.F., S.N., and E.H. helped with AAV construct design and cloning. S.W., R.E.B., and B.T.H. discussed results and wrote the manuscript. Competing interests: The authors declare that they have no competing interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. Additional data related to this paper may be requested from the authors. The plasmid encoding reporter 2A tau constructs can be provided by B.T.H. pending scientific review and a completed material transfer agreement. Requests for the material should be submitted to bhyman{at}mgh.harvard.edu. Additional data are available upon request from susanne.wegmann{at}dzne.de.
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