Transgenic mouse lines ALZ17 and P301S tau were used 6,8
. Mice from line ALZ17, which express the longest human brain tau isoform (441 amino acids) do not exhibit filamentous tau aggregates (Supplementary Information, Fig. S1a
). By contrast, mice from line P301S tau, which express the 383 amino acid human tau isoform with the P301S mutation that causes inherited frontotemporal dementia, develop abundant filamentous tau inclusions (Supplementary Information, Fig. S1a
). Both the 383 and 441 amino acid tau isoforms contain 4 microtubule-binding repeats, but they differ by the presence of 2 alternatively spliced N-terminal inserts of 29 amino acids each 9
To investigate whether aggregation of tau can be transmitted, we injected diluted extracts of brain homogenates from 6 month-old human P301S tau mice into the hippocampus and the overlying cerebral cortex of 3 month-old ALZ17 mice. Prior to injection, the homogenates were analyzed by immunoblotting and immunoelectron microscopy. Human tau protein bands of 55-64 kDa were detected by Western blotting (Supplementary Information, Fig. S1b
). The slowest migrating tau species were immunoreactive with antibody AT100 (Supplementary Information, Fig. S1b
) and other phosphorylation-dependent anti-tau antibodies (not shown). By immunoelectron microscopy, tau filaments were present in the tissue extracts (Supplementary Information, Fig. S1c
). Injection of brain extract from human P301S tau mice induced filamentous tau pathology in ALZ17 mice, as indicated by the appearance of Gallyas-Braak silver staining 10,11
() and the presence of tau filaments by immunoelectron microscopy (). Gallyas-Braak staining was present intracellularly 6, 12 and 15 months after the injection of brain extract (n=5 per group). In addition to silver-positive nerve cell bodies and processes, the injection of brain extract from P301S tau mice resulted in the appearance of immunoreactivity with antibody AT100 (), indicative of tau filaments 6
. In contrast, no silver-positive lesions were observed at corresponding levels of the hippocampus (Supplementary Information, Fig. S2a
) of 18 month-old non-injected ALZ17 mice or in ALZ17 mice 15 months after the injection of brain extract from non-transgenic control mice. ALZ17 animals injected with P301S extract immunodepleted of tau did not reveal any Gallyas- or AT100-positive structures 6 months post-injection (), demonstrating that the presence of tau in the P301S extract was necessary to induce filamentous tauopathy.
Induction of filamentous tau pathology in ALZ17 mice injected with brain extract from mice transgenic for human P301S tau
AT8 immunoreactivity (reflecting tau hyperphosphorylation), but not AT100 staining, was present in the hippocampus of 18 month-old ALZ17 mice, as previously reported 8
. Following the injection of brain extract from human P301S tau mice, AT8 immunoreactivity became more widespread, indicating the promotion of tau hyperphosphorylation (). Filamentous tau pathology in P301S tau-injected ALZ17 mice was induced in different cell types. Silver-positive structures morphologically indistinguishable from those found in human tauopathies were observed in the brains of injected ALZ17 mice (Supplementary Information, Fig. S2b
). They included neurofibrillary tangles (arrows in panels 1 and 2), neuropil threads (arrowheads in panels 1 and 2) and oligodendroglial coiled bodies (arrows in panels 3 and 4). The silver-positive structures were also immunoreactive for phosphorylated tau (Supplementary Information, Fig. S2b panels 2 and 3
). Filaments extracted from injected brains were decorated by phosphorylation-dependent anti-tau antibodies and by antibodies specific for tau isoforms with N-terminal inserts (). It follows that the filaments had formed from the wild-type 441 amino acid human tau isoform expressed in line ALZ17 and were not derived from the injected material. This is also supported by the finding that inclusions in ALZ17 mice injected with P301S brain extract were immunoreactive with antibodies specific for tau with N-terminal inserts (Supplementary Information, Fig. S3
). No silver staining was observed in ALZ17 mice 1 day after the injection of P301S tau brain extract (not shown). No signs of neuronal loss, astrogliosis, inflammation, axonal damage or myelin breakdown were observed in ALZ17 mice 15 months after the injection with P301S tau brain extract when compared to non-injected ALZ17 animals (Supplementary Information, Fig. S4
The induction of filamentous tau in ALZ17 mice was time- and brain region- dependent. Quantitative assessment in the hippocampus revealed a significant increase in the number of silver-positive lesions between 6, 12 and 15 months after injection (). Neuropil threads were most abundant, followed by coiled bodies and neurofibrillary tangles (). The same was true for the cerebral cortex, although fewer silver-positive structures developed there over time ().
Temporal increase in the number of Gallyas-Braak-positive structures at the injection sites (− 2.5 mm from bregma) in ALZ17 mice
Positive silver staining did not remain confined to the injected areas, but spread also to neighbouring brain regions, as visualized in three coronal brain sections encompassing the injection sites and the levels 1.7 mm anterior and 1.3 mm posterior to the injection site (, ). At the injection level, abundant silver staining was present in the hippocampus, fimbria, optic tract and thalamus with fewer abnormal structures in more distant regions, such as medial lemniscus, zona incerta and cerebral peduncle. The hypothalamus was the most distant region where filamentous tau pathology developed (4 mm ventral from the injection sites; an adult mouse brain measures approximately 5.5 mm in height and 13 mm in length). Almost 2 mm anterior to the injection level, filamentous tau pathology was found in the fimbria, thalamus, internal capsule, caudate-putamen, somatosensory cortex, hypothalamus, and the amygdala 15 months after the injection of brain extract from human P301S tau mice (, Supplementary Information, Fig. S5a
). More than 1 mm posterior to the injection level, the cerebral peduncle, hippocampus, superior colliculus, substantia nigra, entorhinal cortex, deep mesencephalic nucleus, and the pontine nuclei exhibited filamentous lesions 15 months after injection (, Supplementary Information, Fig. S5b
). Moderate filamentous tau pathology was observed in some brain regions of the contralateral, non-injected hemisphere (Supplementary Information, Fig. S5c
Spreading of filamentous tau pathology in ALZ17 mice injected with brain extract from mice transgenic for human P301S tau
Semi-quantitative grading of filamentous tau pathology in ALZ17 mice injected with brain extract from mice transgenic for human P301S tau (n=5 for each time point).
To determine which kind of tau species was responsible for the induction of aggregated tau in ALZ17 mice, P301S extracts containing either soluble or insoluble tau were injected into ALZ17 mice. Injection of insoluble tau induced a large number of Gallyas-Braak-positive structures in ALZ17 animals (Supplementary Information, Fig. S6
), similar to what was observed after injection of crude P301S extracts. In addition, there was a similar increase and spreading of fibrillar tau pathology over time (data not shown). Injection of soluble tau induced Gallyas-Braak-positive structures to a much lesser extent (<5%), when compared with insoluble tau (Supplementary Information, Fig. S6
). Thus, it is predominantly insoluble tau species that induce tau aggregation.
We next injected brain extract from human P301S tau mice into non-transgenic control mice. Interestingly, the wild-type mice showed Gallyas-Braak- and AT100-positive threads and coiled bodies (but no NFTs) 6 months () and 12 months after injection (data not shown). They remained confined to the injection site and did not increase in number between 6 and 12 months post-injection (data not shown). Gallyas-Braak-positive structures were stained with anti-murine tau antibody MT1, but not with human tau-specific antibody T14, demonstrating that filamentous tau induced in wild-type mice was made of murine tau (). No tau pathology was observed in sham-lesioned mice (). These results suggest that the expression of human tau in ALZ17 mice was essential for the temporal increase and spreading of filamentous tau pathology after injection of human P301S tau brain extract. However, the presence of a small number of mouse tau filaments in the extract could be responsible for the aggregation of murine tau in wild-type animals and explain the modest amount of filamentous tauopathy. It remains to be clarified to what extent tau expression levels influence spreading.
Induction of filamentous tau pathology in non-transgenic C57BL/6 mice injected with brain extract from mice transgenic for human P301S tau
The present findings show that the intracerebral injection of brain extract from mice with a filamentous tau pathology induces the formation and spreading of silver-positive aggregates made of hyperphosphorylated tau in mice transgenic for human wild-type tau, demonstrating the experimental transmission of tauopathy. During the process leading to Alzheimer's disease, neuronal tau pathology forms in a stereotypical fashion in transentorhinal cortex, from where it appears to spread to hippocampal formation and neocortex 2
, consistent with a uniform biological process. We also show the appearance over time of silver-positive structures at sites that are at a considerable distance from the injection sites in hippocampus and cerebral cortex. Brain regions that develop pathology are connected anatomically to the injection sites or to each other. This, together with the stereotypical appearance of silver-positive structures in defined brain regions over time in all animals studied (), is clearly indicative of the active induction and spreading of pathology rather than the passive diffusion of tau aggregates from the injection sites to more distant regions. The lack of obvious signs of neurodegeneration in ALZ17 mice 15 months after the injection with brain extract from mice transgenic for human P301S tau contrasts with the nerve cell loss that characterizes the P301S tau mice 6
. This suggests that the molecular tau species responsible for transmission and neurotoxicity are not identical. It remains to be seen whether neurodegenerative changes appear at later time points.
A combined neuronal and glial tau pathology is the defining feature of a number of human neurodegenerative diseases characterized by the assembly of wild-type four-repeat tau isoforms into filaments. They include progressive supranuclear palsy, corticobasal degeneration and argyrophilic grain disease 12
. By contrast, in Pick's disease, three-repeat tau isoforms are found in the mostly neuronal inclusions and in Alzheimer's disease, both three- and four-repeat tau isoforms make up neurofibrillary tangles 12
. In addition, the morphologies of tau filaments vary widely in different tauopathies 13
. Together with the findings reported here showing transmission and spreading of filamentous tau pathology, this is reminiscent of mammalian and yeast prions, for which different strains have been described, based on the existence of separate conformers of assembled protein 14
. It is tempting to speculate that distinct tau strains may underlie the pathogenesis of different sporadic tauopathies. This hypothesis can now be tested experimentally by injecting ALZ17 mice with brain extracts from patients with sporadic tauopathies.
From the increase in silver-positive structures over time, it appears likely that nerve cell processes and oligodendrocytes are major sites of filament induction. These findings are comparable to previous work using mouse models of prion diseases, β-amyloid deposition and some peripheral amyloidoses 15-22
. However, unlike the extracellular location of filamentous deposits in those other diseases, tau inclusions are intracellular. Mechanisms must therefore exist by which tau aggregates can either gain access to the inside of cells or activate filament-inducing cascades from the outside. Inside cells, tau aggregates could function as seeds for the ordered assembly of transgenic wild-type human tau into silver- and AT100-positive filamentous deposits. Intracellular filamentous tau pathology could also be induced by extracellular tau aggregates. Previous work has indeed shown potentiation of tau pathology by extracellular β-amyloid aggregates in mice transgenic for mutant human tau 18,19
. However, no such effect was observed in mice transgenic for wild-type human tau 23
It has been suggested that misfolded superoxide dismutase, which causes a subset of familial forms of amyotrophic lateral sclerosis, is secreted from motor neurons 24
. By analogy, spreading of tau pathology may also result from the release of aggregates from affected nerve cells and glial cells. It will be important to investigate whether such mechanisms exist for misfolded tau. The formation of tau aggregates in a single brain cell and their subsequent spreading may therefore be at the origin of sporadic tauopathies. Consequently, the presence of tau aggregates in the extracellular space would be an obligatory step in the events leading to disease. Immunisation strategies may prevent this process 25
The present findings demonstrate the transmission of tauopathy between transgenic mouse lines and describe an experimental system in which to investigate the spreading of pathology and the existence of tau strains. It will be important to identify the molecular tau species capable of inducing transmission, aggregation and spreading. Similar mechanisms may also underlie Parkinson's disease and dementia with Lewy bodies, where α-synuclein pathology appears to spread from brainstem areas to midbrain and neocortex 26
. Furthermore, in Parkinson's disease patients who had undergone transplantation of fetal midbrain neurons, filamentous α-synuclein pathology appeared to spread from affected host tissue to the grafted neurons 27
. Unlike prion diseases 28
, human tauopathies are not believed to be infectious. Experimental model systems of the type described here now make it possible to dissect the similarities and differences between tauopathies and prion diseases.