“Prion‐Like” Templated Misfolding in Tauopathies

Abstract

The soluble microtubule‐associated protein tau forms hyperphosphorylated, insoluble and filamentous inclusions in a number of neurodegenerative diseases referred to as “tauopathies.” In Alzheimer's disease, tau pathology develops in a stereotypical manner, with the first lesions appearing in the locus coeruleus and entorhinal cortex, from where they appear to spread to the hippocampus and neocortex. Propagation of tau pathology is also a characteristic of argyrophilic grain disease, where the tau lesions spread throughout the limbic system. Significantly, isoform composition and morphology of tau filaments can differ between tauopathies, suggesting the existence of distinct tau strains. Extensive experimental findings indicate that prion‐like mechanisms underly the pathogenesis of tauopathies.

Tau Protein

In 1975, a protein was isolated from brain and named tau because of its ability to induce microtubule formation 100. Tau is a natively unfolded microtubule‐associated protein that binds microtubules and that may play a role in their assembly and stabilization 36. In nerve cells, tau is concentrated in axons 8, but it may also have a physiological function in dendrites 52. In the 1980s and early 1990s, immunohistochemistry, electron microscopy, biochemistry and molecular biology were used to establish that the paired helical and straight filaments of Alzheimer's disease (AD) are composed of all human brain tau isoforms in a hyperphosphorylated state 16, 39, 41, 44, 45, 57, 63, 103. Six tau isoforms are expressed in adult human brain and are derived by alternative mRNA splicing from a single gene (MAPT) located on chromosome 17q21.31 3, 40, 69 (Figure 1). They differ from each other by the presence or absence of a 29‐ or 58‐amino insert in the amino‐terminal half and by the inclusion, or not, of a 31‐amino acid repeat encoded by exon 10 of MAPT in the carboxy‐terminal half of the protein. The inclusion of exon 10 results in the production of three isoforms with four repeats each and its exclusion in the production of an additional three isoforms with three repeats each. The repeats and some adjoining sequences constitute the microtubule‐binding domains of tau 43, with four‐repeat tau being better at promoting microtubule assembly than tau with three repeats 38. Residues S214‐E372 of tau, corresponding to the region upstream of the repeats and the repeats themselves, bind tightly to taxol‐stabilized microtubules 30.

Figure 1.

Schematic representation of MAPT and the six tau isoforms expressed in adult human brain. Human MAPT consists of 16 exons (E). Alternative mRNA splicing of E2 (pink), E3 (green) and E10 (yellow) gives rise to the six tau isoforms (352–441 amino acids). E1, E4, E5, E7, E9, E11, E12 and E13 (blue) are constitutively spliced exons. E0, which is part of the promoter, and E14, are non‐coding (white). E6 and E8 (violet), as well as E4a (orange) are not transcribed in human brain. The core regions of the repeats of tau are shown as black bars. Three isoforms have four repeats each (4R hTau) and three isoforms have three repeats each (3R hTau). Similar levels of 4R and 3R tau isoforms are expressed in normal adult human brain. The exons and introns are not drawn to scale.

Filamentous tau is hyperphosphorylated and unable to interact with microtubules 15, 110. Hyperphosphorylation promotes the dissociation of tau from microtubules and may lead to its redistribution from the axonal to the somatodendritic compartment. Antibodies, such as AT8, visualize hyperphosphorylated tau before and after its aggregation into filaments 36. Filamentous tau can be specifically detected by the phosphorylation‐dependent antibody AT100 and by silver staining. Many phosphorylation sites are known, as are candidate protein kinases and phosphatases 36, 48. Proline‐directed protein kinases, protein kinases that phosphorylate the KxGS motifs in the repeats, some tyrosine kinases and protein phosphatase 2A have all been implicated in the phosphorylation and dephosphorylation of tau. In human tauopathies, tau may first misfold, making it a better substrate for protein kinases and a less good substrate for protein phosphatases, resulting in its hyperphosphorylation and increased likelihood of aggregation. Although hyperphosphorylation is a major early characteristic of abnormal tau, other post‐translational modifications have also been described. They include tau acetylation 20, 51, 67, glycation 61, 87, O‐GlcNAcylation 5, nitration 79, ubiquitination 21, 68, sumoylation 28, prolyl isomerization 65 and truncation 34, 104.

Neurodegenerative Diseases with Tau Pathology

Tau aggregates are present in a large number of neurodegenerative diseases known as the “tauopathies” 36, 90 (Table 1). They include AD, tangle‐only dementia (TD), argyrophilic grain disease (AGD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), Pick's disease (PiD), as well as frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP‐17T), the latter being caused by mutations in MAPT  49, 75, 91. Although these diseases have lesions in common, such as gross brain atrophy, nerve cell loss, gliosis, superficial spongiosis and ballooned neurons, they also tend to exhibit distinct tau pathologies. Thus, in AD and TD, both three‐ and four‐repeat tau isoforms make up the neuronal inclusions 39, 72, whereas in PiD tau, isoforms with three repeats predominate in the neuronal deposits 27. The assembly of four‐repeat tau into filaments is a characteristic of PSP, CBD and AGD 32, 58, 93, 96. Although PSP and CBD are four‐repeat tauopathies, their tau cleavage products vary in size. Detergent‐insoluble cleaved tau from PSP migrates as a single band of 33 kDa, whereas that of CBD migrates as a doublet of 37 kDa 4. Filaments from human tauopathy brains also exhibit a range of morphologies 23 and specific conformers of aggregated tau may give rise to distinct tauopathies.

Table 1.

Diseases with tau inclusions

Alzheimer disease

Amyotrophic lateral sclerosis/parkinsonism‐dementia complex

Argyrophilic grain disease

Chronic traumatic encephalopathy

Corticobasal degeneration

Diffuse neurofibrillary tangles with calcification

Down syndrome

Familial British dementia

Familial Danish dementia

Frontotemporal dementia and parkinsonism linked to chromosome 17 caused by MAPT mutations

Frontotemporal lobar degeneration (caused by C9ORF72 mutations)

Gerstmann–Sträussler–Scheinker disease

Guadeloupean parkinsonism

Myotonic dystrophy

Neurodegeneration with brain iron accumulation

Niemann–Pick disease, type C

Non‐Guamanian motor neuron disease with neurofibrillary tangles

Pick disease

Post‐encephalitic parkinsonism

Prion protein cerebral amyloid angiopathy

Progressive subcortical gliosis

Progressive supranuclear palsy

SLC9A6‐related mental retardation

Subacute sclerosing panencephalitis

Tangle‐only dementia

White matter tauopathy with globular glial inclusions

Besides the involvement of specific tau isoforms and the presence of filaments with distinct morphologies, the cellular tau pathologies can also be a characteristic. Thus, in AD and TD, tau inclusions occur in the form of neurofibrillary tangles (NFTs) and neuropil threads (NTs). NFTs are located in the somatodendritic compartment, whereas NTs are found in distal axons and dendrites. In AGD, abundant argyrophilic grains in neuronal processes, pre‐tangle neurons in limbic areas, as well as glial tau inclusions in astrocytes and oligodendrocytes make up the hallmark lesions 10, 94. PSP brains are characterized by neuronal tau inclusions known as globose‐type NFTs and NTs 77, as well as by glial changes in the form of tufted astrocytes and oligodendroglial coiled bodies 70, 109. CBD brains show intracytoplasmic pathological tau in NTs, pre‐tangle neurons or small NFTs, as well as astrocytic plaques and coiled bodies 31, 56, 95. Pick bodies are mainly present in neocortical, hippocampal and subcortical nerve cells of patients with PiD 78. Neuropathologically, FTDP‐17T presents with severe nerve cell loss, astrocytic gliosis and spongiosis, with filamentous tau inclusions in nerve cells or in both nerve cells and glial cells. Depending on the MAPT mutations, the inclusions are composed predominantly of three repeats, four repeats or a mixture of all six brain tau isoforms 35. As of February 2013, 49 MAPT mutations have been described.

During the course of AD and AGD, tau inclusions appear to propagate through the brain in a stereotypical fashion, thus providing the basis for reliable disease staging. In human brain, tau inclusions appear at a younger age than Aβ plaques 13, 14. Sparse tau pathology has been described in the brains of a substantial proportion of individuals under the age of 30. Hyperphosphorylated tau accumulated first in the locus coeruleus, from where the pathology spread to the entorhinal cortex and other brain regions. This differential distribution underlies the six “Braak stages” of AD. Stages I and II correspond to the presence of NFTs in transentorhinal and entorhinal cortex and are not associated with clinical symptoms. A more pronounced involvement of transentorhinal and entorhinal cortex and the formation of NFTs in the hippocampus are a characteristic of stages III and IV, which can be associated with the first clinical symptoms. Stages V and VI correspond to the extensive spreading of NFTs to isocortical association areas. Patients with Braak stages V and VI are severely demented and meet the neuropathological criteria of AD. Many tau inclusions survive the death of affected nerve cells as extracellular or ghost tangles. In AGD, the earliest accumulation of tau inclusions is present in the ambient gyrus (stage I), from where the pathology propagates to the anterior and posterior medial temporal lobe (stage II), followed by septum, insular cortex and anterior cingulate gyrus (stage III). Stage III is associated with a clinical diagnosis of dementia 80.

Experimental Transmission of Tauopathy

The characteristics of the human tauopathies are consistent with the existence of distinct tau conformers (or strains). Moreover, the propagation of tau pathology during the clinical course of tauopathies points to the existence of mechanisms for the intercellular transfer of tau aggregates. This notion has been substantiated through the identification and characterization of the cell‐to‐cell propagation of tau inclusions 19, 33.

We used mouse lines transgenic for single isoforms of human wild‐type (line ALZ17) and mutant (line P301S) tau to investigate the induction and propagation of tau pathology 2, 19, 76. We injected brain homogenates from P301S mice with numerous AT100‐ and silver‐positive tau inclusions into the hippocampus and overlying cerebral cortex of ALZ17 mice, which do not develop filamentous tauopathy. As a result, wild‐type human tau assembled in ALZ17 mice into filaments in neurons and oligodendrocytes in the form of NFTs, NTs and coiled bodies (Figure 2). Strikingly, the induction of filamentous tau was not restricted to the injection sites, but progressed over time to both neighboring and more distant anatomically connected brain regions. Signs of neurodegeneration were not observed for up to 18 months after the injection of P301S tau brain homogenates. This supports the suggestion that the molecular tau species responsible for propagation and toxicity are different 19.

Figure 2.

Tau transgenic mouse models and experimental induction of filamentous tauopathy. A. Left: Mice expressing the 383 amino acid 4R human tau with the P301S mutation under the control of the murine Thy1 promoter develop abundant hyperphosphorylated (AT8‐immunoreactive) and Gallyas–Braak silver‐positive filamentous tau inclusions in various brain regions, including the brainstem, from which extracts were prepared for injection into the brains of ALZ17 mice. Right: ALZ17 mice express the 441‐amino acid 4R human tau isoform under the control of the murine Thy1 promoter. These mice develop tau that is immunoreactive with the phosphorylation‐dependent antibody AT8, but Gallyas–Braak silver‐negative as shown here for the hippocampus. These mice also lack tau filaments. The sections were counterstained with hematoxylin. Scale bar: 50 μm (the same magnification in all panels). B. Staining of the hippocampal CA3 region from 18‐month‐old ALZ17 mice with AT8, Gallyas–Braak silver and phosphorylation‐dependent anti‐tau antibody AT100, 15 months after no injection (upper panel) or after the injection with brain extract from 6‐month‐old P301S tau mice (lower panel). The injection of brain extracts induced the formation of AT100‐ and Gallyas–Braak silver‐positive filamentous inclusions made of wild‐type human tau. No such inclusions were found in ALZ17 mice injected with extracts from control mice 19. The sections were counterstained with hematoxylin. Scale bar: 50 μm (the same magnification in all panels). C. Different types of filamentous tau pathology in ALZ17 brains after the injection with brain extracts from mice transgenic for human P301S tau: Gallyas–Braak silver impregnation shows a neurofibrillary tangle (left), neuropil threads (middle, arrows) and coiled bodies (right, arrows). The sections were counterstained with hematoxylin. Scale bar: 50 μm (the same magnification in all panels).

These findings were confirmed and extended by additional work in vivo. One study reported the induction and propagation of tau pathology after the injection of tau oligomers from AD brain into wild‐type mice 59. Silver‐positive staining was present in the hippocampus, as well as in the cerebral cortex, corpus callosum and hypothalamus. These findings are consistent with the view that tau oligomers are the major molecular species responsible for tau induction and spreading. However, the purity of the tau species immunoprecipitated from AD brain homogenates remains to be established more fully. Originally, we showed that the inclusion‐inducing activity resided in the insoluble tau fraction of P301S tau brain 19. This conclusion was supported by the demonstration that tau filaments assembled from human mutant recombinant protein promoted the formation of tau inclusions in presymptomatic mice transgenic for human mutant P301S tau 50. After the injection of synthetic tau filaments into the hippocampus, tau inclusions also formed in the entorhinal cortex and the contralateral (non‐injected) hippocampus; after injection into the striatum, tau inclusions formed in the substantia nigra, the thalamus and the corpus callosum. We obtained similar results after the injection of tau filaments assembled from recombinant human P301S tau in the presence of heparin into the hippocampus and overlying cerebral cortex of 3‐month‐old homozygous mice transgenic for human mutant P301S tau. Abundant silver‐positive tau inclusions had formed at the injection sites 4 weeks after the injection (Figure 3). These findings are compatible with the view that tau filaments are sufficient for recruiting and converting soluble tau into pathological inclusions. However, one must also bear in mind that the assembly mixtures used for injection were probably heterogeneous at the molecular level. It remains to be seen if brain extracts were more effective than recombinant assembled tau at promoting assembly, as has been described for other amyloidogenic proteins 29. In addition, transgenic mice expressing human mutant P301L tau in some entorhinal cortical neurons were found to develop tau inclusions not only in parts of the entorhinal cortex and the subiculum but also in the dentate gyrus and in layers CA1 and CA3 of the hippocampus, consistent with their trans‐synaptic spread 26, 64. The possibility that the tau inclusions that formed outside the entorhinal cortex may have been the result of leaky transgene expression was reported to have been ruled out. It follows that aggregated human tau must have been able to convert soluble mouse tau into filaments.

Figure 3.

Injection of synthetic tau filaments into the hippocampus induces the formation of tau inclusions in mice transgenic for human mutant P301S tau. A. Gallyas–Braak silver stained section of the hippocampus from a 4‐month‐old homozygous mouse transgenic for human mutant P301S tau that had been injected with 7.5 μg assembled recombinant human P301S tau (four‐repeat isoform lacking amino‐terminal inclusions) at the age of 3 months. For assembly, recombinant human P301S tau (3 mg/mL) was incubated with heparin (400 μg/mL) for 48 h at 37°C. B. Gallyas–Braak silver stained section of the hippocampus from a 4‐month‐old non‐injected mouse transgenic for human mutant P301S tau. Scale bar: 50 μm.

Monomeric tau has been detected in the interstitial fluid of the brain, suggesting that it was released, despite the lack of a signal sequence 108. The secretion of non‐aggregated tau has also been described in cultured cells 53, 81, where extracellular tau was reported to be neurotoxic because of an increase in intracellular calcium levels following the activation of some muscarinic cholinergic receptors 42. A link may exist between the secretion of monomeric tau and increased tau levels in cerebrospinal fluid. However, it is at present unclear how these processes may relate to the propagation of tau aggregates.

The intercellular transfer of tau inclusions has also been demonstrated in cultured cells 33, 46, 54, 82, 106. Filaments made of recombinant tau and tau filaments from AD brain were taken up by cultured cells and induced the aggregation of soluble cytoplasmic tau. The aggregates co‐localized with dextran, a marker of fluid phase endocytosis, but not with cholera toxin B, a marker of lipid rafts, suggesting the involvement of endocytic pathways. Aggregates could also transfer between cells in a co‐culture system, consistent with their release and uptake. These experimental systems lend themselves to the elucidation of the molecular mechanisms underlying the propagation of tau aggregates.

Therapeutical Implications

The events leading from soluble, monomeric to insoluble, filamentous tau are central to the human tauopathies 36. Inhibiting aggregation and/or disassembling tau aggregates therefore constitute important therapeutical approaches. The repeat region of tau makes up the core of the tau filament 40, 103, 104 and the third tau repeat, whose amino‐terminus consists of the hexapeptide ³⁰⁶VQIVYK³¹¹, is central for assembly 24. An all d‐amino acid peptide was produced, which inhibited tau filament formation 85. Small‐molecule inhibitors of tau filament assembly have also been described 17, 22, 92, including a number of porphyrins, polyphenols and phenothiazines. The porphyrin phthalocyanine tetrasulphonate interfered with tau filament formation by causing the production of soluble, non‐toxic oligomers that lacked significant β‐structure 1. A polyphenolic grape seed extract reduced the formation of tau inclusions in a mouse model of tauopathy 99. The phenothiazine methylene blue was the first compound shown to inhibit the aggregation of tau 102. It reduced the levels of soluble tau and improved cognitive function in transgenic mice 73. In a phase II clinical trial of patients with mild to moderate AD, methylene blue improved cognition 101. A phase III trial is ongoing.

Hyperphosphorylation seems to be essential for tau‐mediated neurodegeneration. As a result, the inhibition of tau kinases and the activation of tau phosphatases are therapeutic approaches. The number of hyperphosphorylated sites in tau is large, suggesting the involvement of multiple protein kinases. The non‐specific protein kinase inhibitor K252a reduced the levels of soluble aggregated hyperphosphorylated tau and improved motor symptoms in a mouse line transgenic for human mutant P301L tau 60. Inhibitors of the proline‐directed glycogen synthase kinase‐3 (GSK‐3) had a similar effect in mouse models 71. However, a short‐term trial of lithium chloride, a non‐specific inhibitor of GSK‐3, in patients with AD, was not successful 47. The same was true of tideglusib (NP‐12), a specific GSK‐3 inhibitor, in patients with AD (Press release of 11 October 2012: Noscira announces results from ARGO phase IIb trial of tideglusib for the treatment of Alzheimer's disease).

PP2A is the major tau phosphatase in the brain 37. It is a heterotrimeric protein consisting of a catalytic (C), a scaffolding (A) and a regulatory (B) subunit. There are multiple genes for each subunit. In brain, isoform ACB55α is the major tau phosphatase 88, 107. Folic acid, whose reduction in AD contributes to elevated homocysteine, promotes the methylation of PP2Ac 89, resulting in increased phosphatase activity 105. The anti‐diabetes drug metformin has been reported to decrease tau phosphorylation through increased PP2A activity 55. Moreover, chronic low doses of sodium selenate reduced tau phosphorylation in mouse models of tauopathy through an increase in PP2A activity 98.

O‐GlcNAcylation, by which N‐acetylglucosamine is attached to serine and threonine residues, is another post‐translational modification of tau 5. Treatment with Thiamet G, an inhibitor of the enzyme responsible for the hydrolysis of O‐linked N‐acetylglucosamine, reduced tau aggregation and increased nerve cell survival in a mouse model of tauopathy through a reduction in the oligomerization of tau 111.

As is the case of other amyloids, the assembly of tau into filaments is energetically unfavorable and concentration‐dependent. A reduction in the levels of tau is therefore an attractive therapeutical approach that may be achieved directly through antisense or RNAi approaches, or indirectly through decreased tau expression and increased tau clearance. A partial reduction in tau levels in mice appears to be well tolerated. Soluble tau is degraded by the ubiquitin‐proteasome system 25, with the carboxy‐terminus of heat shock protein (Hsp) 70‐interacting protein (CHIP) and Hsp90 being important 66, 74. The ubiquitin ligase CHIP binds to phosphorylated tau and is required for its proteasomal degradation. USP14, a deubiquitinating enzyme that inhibits the degradation of ubiquitinated proteins, increased soluble tau levels. Conversely, a small‐molecule inhibitor of USP14 enhanced tau degradation 62. Stimulated clearance of tau aggregates is a complementary approach. Aggregates are probably not accessible to the ubiquitin/proteasome, but may be degraded by the autophagy/lysosome system. In a mouse line transgenic for human mutant tau, trehalose, which activates autophagy in an mTOR‐independent manner 83, reduced the number of tau inclusions and improved nerve cell survival 84.

If tau aggregates propagate between cells, they may pass through an obligate extracellular stage. It follows that the removal of extracellular tau aggregates by specific antibodies may constitute a mechanism‐based therapy. To date, seven studies using either active or passive immunization against tau have reported beneficial effects in transgenic mouse models of tauopathy 6, 7, 9, 11, 12, 18, 97. Active immunization may carry an intrinsic risk of causing tauopathy. Active immunization used tau phosphopeptides as antigens, whereas passive immunization studies used antibodies against either phosphorylated or non‐phosphorylated tau 86.

Conclusion

The mechanisms underlying the stereotypical spreading of tau pathology in late‐onset AD and AGD remain to be discovered. For many years, tau pathology was believed to form in a cell autonomous manner. As discussed here, numerous experimental studies have now established that tau aggregates can be released and taken up by neighboring cells. This is consistent with tau aggregation originating in a small number of cells, from where it spreads in a non‐cell autonomous manner. Similar mechanisms may operate in AD, AGD and other human tauopathies, such as PSP, CBD and PiD. The phenotypic and pathogenic variations between tauopathies may be explained by the existence of distinct tau conformers (or strains).

Conflict of interest

The authors declare that they have no conflict of interest related to this article.