Cryo-EM structures of tau filaments from the brains of mice transgenic for human mutant P301S Tau.

Mice transgenic for human mutant P301S tau are widely used as models for human tauopathies. They develop neurodegeneration and abundant filamentous inclusions made of human mutant four-repeat tau. Here we used electron cryo-microscopy (cryo-EM) to determine the structures of tau filaments from the brains of Tg2541 and PS19 mice. Both lines express human P301S tau (0N4R for Tg2541 and 1N4R for PS19) on mixed genetic backgrounds and downstream of different promoters (murine Thy1 for Tg2541 and murine Prnp for PS19). The structures of tau filaments from Tg2541 and PS19 mice differ from each other and those of wild-type tau filaments from human brains. The structures of tau filaments from the brains of humans with mutations P301L, P301S or P301T in MAPT are not known. Filaments from the brains of Tg2541 and PS19 mice share a substructure at the junction of repeats 2 and 3, which comprises residues I297-V312 of tau and includes the P301S mutation. The filament core from the brainstem of Tg2541 mice consists of residues K274-H329 of tau and two disconnected protein densities. Two non-proteinaceous densities are also in evidence. The filament core from the cerebral cortex of line PS19 extends from residues G271-P364 of tau. One strong non-proteinaceous density is also present. Unlike the tau filaments from human brains, the sequences following repeat 4 are missing from the cores of tau filaments from the brains of Tg2541 and PS19 mice.

Cryo-EM structures of amyloid-ß filaments with the Arctic mutation (E22G) from human and mouse brains.

The Arctic mutation, encoding E693G in the amyloid precursor protein (APP) gene [E22G in amyloid-ß (Aß)], causes dominantly inherited Alzheimer's disease. Here, we report the high-resolution cryo-EM structures of Aß filaments from the frontal cortex of a previously described case (AßPParc1) with the Arctic mutation. Most filaments consist of two pairs of non-identical protofilaments that comprise residues V12-V40 (human Arctic fold A) and E11-G37 (human Arctic fold B). They have a substructure (residues F20-G37) in common with the folds of type I and type II Aß42. When compared to the structures of wild-type Aß42 filaments, there are subtle conformational changes in the human Arctic folds, because of the lack of a side chain at G22, which may strengthen hydrogen bonding between mutant Aß molecules and promote filament formation. A minority of Aß42 filaments of type II was also present, as were tau paired helical filaments. In addition, we report the cryo-EM structures of Aß filaments with the Arctic mutation from mouse knock-in line AppNL-G-F. Most filaments are made of two identical mutant protofilaments that extend from D1 to G37 (AppNL-G-F murine Arctic fold). In a minority of filaments, two dimeric folds pack against each other in an anti-parallel fashion. The AppNL-G-F murine Arctic fold differs from the human Arctic folds, but shares some substructure.

Cryo-EM structures of amyloid-ß 42 filaments from human brains.

Filament assembly of amyloid-ß peptides ending at residue 42 (Aß42) is a central event in Alzheimer's disease. Here, we report the cryo­electron microscopy (cryo-EM) structures of Aß42 filaments from human brains. Two structurally related S-shaped protofilament folds give rise to two types of filaments. Type I filaments were found mostly in the brains of individuals with sporadic Alzheimer's disease, and type II filaments were found in individuals with familial Alzheimer's disease and other conditions. The structures of Aß42 filaments from the brain differ from those of filaments assembled in vitro. By contrast, in AppNL-F knock-in mice, Aß42 deposits were made of type II filaments. Knowledge of Aß42 filament structures from human brains may lead to the development of inhibitors of assembly and improved imaging agents.

Increase in Tau Pathology in P290S Mapt Knock-In Mice Crossed with App NL-G-F Mice.

Alzheimer's Disease (AD) is characterized by the pathologic assembly of amyloid ß (Aß) peptide, which deposits into extracellular plaques, and tau, which accumulates in intraneuronal inclusions. To investigate the link between Aß and tau pathologies, experimental models featuring both pathologies are needed. We developed a mouse model featuring both tau and Aß pathologies by knocking the P290S mutation into murine Mapt and crossing these Mapt P290S knock-in (KI) mice with the App NL-G-F KI line. Mapt P290S KI mice developed a small number of tau inclusions, which increased with age. The amount of tau pathology was significantly larger in App NL-G-F xMapt P290S KI mice from 18 months of age onward. Tau pathology was higher in limbic areas, including hippocampus, amygdala, and piriform/entorhinal cortex. We also observed AT100-positive and Gallyas-Braak-silver-positive dystrophic neurites containing assembled filamentous tau, as visualized by in situ electron microscopy. Using a cell-based tau seeding assay, we showed that Sarkosyl-insoluble brain extracts from both 18-month-old Mapt P290S KI and App NL-G-F xMapt P290S KI mice were seed competent, with brain extracts from double-KI mice seeding significantly more than those from the Mapt P290S KI mice. Finally, we showed that App NL-G-F xMapt P290S KI mice had neurodegeneration in the piriform cortex from 18 months of age. We suggest that App NL-G-F xMapt P290S KI mice provide a good model for studying the interactions of aggregation-prone tau, Aß, neuritic plaques, neurodegeneration, and aging.

Assembly of α-synuclein and neurodegeneration in the central nervous system of heterozygous M83 mice following the peripheral administration of α-synuclein seeds.

Peripheral administration (oral, intranasal, intraperitoneal, intravenous) of assembled A53T α-synuclein induced synucleinopathy in heterozygous mice transgenic for human mutant A53T α-synuclein (line M83). The same was the case when cerebellar extracts from a case of multiple system atrophy with type II α-synuclein filaments were administered intraperitoneally, intravenously or intramuscularly. We observed abundant immunoreactivity for pS129 α-synuclein in nerve cells and severe motor impairment, resulting in hindlimb paralysis and shortened lifespan. Filaments immunoreactive for pS129 α-synuclein were in evidence. A 70% loss of motor neurons was present five months after an intraperitoneal injection of assembled A53T α-synuclein or cerebellar extract with type II α-synuclein filaments from an individual with a neuropathologically confirmed diagnosis of multiple system atrophy. Microglial cells changed from a predominantly ramified to a dystrophic appearance. Taken together, these findings establish a close relationship between the formation of α-synuclein inclusions in nerve cells and neurodegeneration, accompanied by a shift in microglial cell morphology. Propagation of α-synuclein inclusions depended on the characteristics of both seeds and transgenically expressed protein.

α-Synuclein filaments from transgenic mouse and human synucleinopathy-containing brains are major seed-competent species.

Assembled α-synuclein in nerve cells and glial cells is the defining pathological feature of neurodegenerative diseases called synucleinopathies. Seeds of α-synuclein can induce the assembly of monomeric protein. Here, we used sucrose gradient centrifugation and transiently transfected HEK 293T cells to identify the species of α-synuclein from the brains of homozygous, symptomatic mice transgenic for human mutant A53T α-synuclein (line M83) that seed aggregation. The most potent fractions contained Sarkosyl-insoluble assemblies enriched in filaments. We also analyzed six cases of idiopathic Parkinson's disease (PD), one case of familial PD, and six cases of multiple system atrophy (MSA) for their ability to induce α-synuclein aggregation. The MSA samples were more potent than those of idiopathic PD in seeding aggregation. We found that following sucrose gradient centrifugation, the most seed-competent fractions from PD and MSA brains are those that contain Sarkosyl-insoluble α-synuclein. The fractions differed between PD and MSA, consistent with the presence of distinct conformers of assembled α-synuclein in these different samples. We conclude that α-synuclein filaments are the main driving force for amplification and propagation of pathology in synucleinopathies.

Silver staining (Campbell-Switzer) of neuronal α-synuclein assemblies induced by multiple system atrophy and Parkinson's disease brain extracts in transgenic mice.

Synucleinopathies [Parkinson's disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA)] share filamentous α-synuclein assemblies in nerve cells and glial cells. We compared the abilities of brain extracts from MSA and PD patients to induce neuronal α-synuclein assembly and neurodegeneration following intracerebral injection in heterozygous mice transgenic for human mutant A53T α-synuclein. MSA extracts were more potent than PD extracts in inducing α-synuclein assembly and in causing neurodegeneration. MSA assemblies were Campbell-Switzer- and Gallyas-silver-positive, whereas PD assemblies were only Campbell-Switzer-positive, in confirmation of previous findings. However, induced α-synuclein inclusions were invariably Campbell-Switzer-positive and Gallyas-negative, irrespective of whether MSA or PD brain extracts were injected. The α-synuclein inclusions of non-injected homozygous mice transgenic for A53T α-synuclein were also Campbell-Switzer-positive and Gallyas-negative. These findings demonstrate that transgene expression and its intracellular environment dominated over the silver staining properties of the conformers of assembled α-synuclein.

Assembly of transgenic human P301S Tau is necessary for neurodegeneration in murine spinal cord.

A pathological pathway leading from soluble monomeric to insoluble filamentous Tau is characteristic of many human neurodegenerative diseases, which also exhibit dysfunction and death of brain cells. However, it is unknown how the assembly of Tau into filaments relates to cell loss. To study this, we first used a mouse line transgenic for full-length human mutant P301S Tau to investigate the temporal relationship between Tau assembly into filaments, assessed using anti-Tau antibody AT100, and motor neuron numbers, in the lumbar spinal cord. AT100 immunoreactivity preceded nerve cell loss. Murine Tau did not contribute significantly to either Tau aggregation or neurodegeneration. To further study the relevance of filament formation for neurodegeneration, we deleted hexapeptides 275VQIINK280 and 306VQIVYK311, either singly or in combination, from human 0N4R Tau with the P301S mutation. These hexapeptides are essential for the assembly of Tau into filaments. Homozygous mice transgenic for P301S Tau with the hexapeptide deletions, which expressed Tau at a similar level to the heterozygous line transgenic for P301S Tau, had a normal lifespan, unlike mice from the P301S Tau line. The latter had significant levels of sarkosyl-insoluble Tau in brain and spinal cord, and exhibited neurodegeneration. Mice transgenic for P301S Tau with the hexapeptide deletions failed to show significant levels of sarkosyl-insoluble Tau or neurodegeneration. Recombinant P301S Tau with the hexapeptide deletions failed to form ß-sheet structure and filaments following incubation with heparin. Taken together, we conclude that ß-sheet assembly of human P301S Tau is necessary for neurodegeneration in transgenic mice.

A novel in vivo model of tau propagation with rapid and progressive neurofibrillary tangle pathology: the pattern of spread is determined by connectivity, not proximity.

Intracellular inclusions composed of hyperphosphorylated filamentous tau are a hallmark of Alzheimer's disease, progressive supranuclear palsy, Pick's disease and other sporadic neurodegenerative tauopathies. Recent in vitro and in vivo studies have shown that tau aggregates do not only seed further tau aggregation within neurons, but can also spread to neighbouring cells and functionally connected brain regions. This process is referred to as 'tau propagation' and may explain the stereotypic progression of tau pathology in the brains of Alzheimer's disease patients. Here, we describe a novel in vivo model of tau propagation using human P301S tau transgenic mice infused unilaterally with brain extract containing tau aggregates. Infusion-related neurofibrillary tangle pathology was first observed 2 weeks post-infusion and increased in a stereotypic, time-dependent manner. Contralateral and anterior/posterior spread of tau pathology was also evident in nuclei with strong synaptic connections (efferent and afferent) to the site of infusion, indicating that spread was dependent on synaptic connectivity rather than spatial proximity. This notion was further supported by infusion-related tau pathology in white matter tracts that interconnect these regions. The rapid and robust propagation of tau pathology in this model will be valuable for both basic research and the drug discovery process.

"Prion-like" templated misfolding in tauopathies.

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.

Stimulation of autophagy reduces neurodegeneration in a mouse model of human tauopathy.

The accumulation of insoluble proteins is a pathological hallmark of several neurodegenerative disorders. Tauopathies are caused by the dysfunction and aggregation of tau protein and an impairment of cellular protein degradation pathways may contribute to their pathogenesis. Thus, a deficiency in autophagy can cause neurodegeneration, while activation of autophagy is protective against some proteinopathies. Little is known about the role of autophagy in animal models of human tauopathy. In the present report, we assessed the effects of autophagy stimulation by trehalose in a transgenic mouse model of tauopathy, the human mutant P301S tau mouse, using biochemical and immunohistochemical analyses. Neuronal survival was evaluated by stereology. Autophagy was activated in the brain, where the number of neurons containing tau inclusions was significantly reduced, as was the amount of insoluble tau protein. This reduction in tau aggregates was associated with improved neuronal survival in the cerebral cortex and the brainstem. We also observed a decrease of p62 protein, suggesting that it may contribute to the removal of tau inclusions. Trehalose failed to activate autophagy in the spinal cord, where it had no impact on the level of sarkosyl-insoluble tau. Accordingly, trehalose had no effect on the motor impairment of human mutant P301S tau transgenic mice. Our findings provide direct evidence in favour of the degradation of tau aggregates by autophagy. Activation of autophagy may be worth investigating in the context of therapies for human tauopathies.

Detection of filamentous tau inclusions by the fluorescent Congo red derivative FSB [(trans,trans)-1-fluoro-2,5-bis(3-hydroxycarbonyl-4-hydroxy)styrylbenzene].

Filamentous inclusions made of the microtubule-associated protein tau in a hyperphosphorylated state are a defining feature of a large number of human neurodegenerative diseases. Here we show that (trans,trans)-1-fluoro-2,5-bis(3-hydroxycarbonyl-4-hydroxy)styrylbenzene (FSB), a fluorescent Congo red derivative, labels tau inclusions in tissue sections from a mouse line transgenic for human P301S tau and in cases of familial frontotemporal dementia and sporadic Pick's disease. Labelling by FSB required the presence of tau filaments. More importantly, tau inclusions in the spinal cord of human P301S tau transgenic mice were labelled following a single intravenous injection of FSB. These findings indicate that FSB can be used to detect filamentous tau in vivo.

Analysis of tau phosphorylation and truncation in a mouse model of human tauopathy.

Recent evidence has suggested that truncation of tau protein at the caspase cleavage site D421 precedes hyperphosphorylation and may be necessary for the assembly of tau into filaments in Alzheimer's disease and other tauopathies. Here we have investigated the time course of the appearance of phosphorylated and truncated tau in the brain and spinal cord of mice transgenic for mutant human P301S tau protein. This mouse line recapitulates the essential molecular and cellular features of the human tauopathies, including tau hyperphosphorylation, tau filament formation, and neurodegeneration. Soluble tau was strongly phosphorylated at 1 to 6 months of age. Low levels of phosphorylated, sarkosyl-insoluble tau were detected at 2 months, with a steady increase up to 6 months of age. Tau truncated at D421 was detected at low levels in Tris-soluble and detergent-soluble tau at 3 to 6 months of age. By immunoblotting, it was not detected in sarkosyl-insoluble tau. However, by immunoelectron microscopy, a small percentage of tau in filaments from brain and spinal cord of transgenic mice was truncated at D421. Similar findings were obtained using dispersed filaments from Alzheimer's disease and FTDP-17 brains. The late appearance and low abundance of tau ending at D421 indicate that it is unlikely that truncation at this site is necessary for the assembly of tau into filaments.

Cell-cycle markers in a transgenic mouse model of human tauopathy: increased levels of cyclin-dependent kinase inhibitors p21Cip1 and p27Kip1.

Recent evidence has suggested that an abnormal reactivation of the cell cycle may precede and cause the hyperphosphorylation and filament formation of tau protein in Alzheimer's disease and other tauopathies. Here we have analyzed the expression and/or activation of proteins involved in cell-cycle progression in the brain and spinal cord of mice transgenic for mutant human P301S tau protein. This mouse line recapitulates the essential molecular and cellular features of the human tauopathies, including hyperphosphorylation and filament formation of tau protein. None of the activators and co-activators of the cell cycle tested were overexpressed or activated in 5-month-old transgenic mice when compared to controls. By contrast, the levels of cyclin-dependent kinase inhibitors p21Cip1 and p27Kip1 were increased in brain and spinal cord of transgenic mice. Both inhibitors accumulated in the cytoplasm of nerve cells, the majority of which contained inclusions made of hyperphosphorylated tau protein. A similar staining pattern for p21Cip1 and p27Kip1 was also present in the frontal cortex from a case of FTDP-17 with the P301L tau mutation. Thus, reactivation of the cell cycle was not involved in tau hyperphos-phorylation and filament formation, consistent with expression of p21Cip1 and p27Kip1 in tangle-bearing nerve cells.

Folding and stability of a primitive protein.

We have previously attempted to simulate domain creation in early protein evolution by recombining polypeptide segments from non-homologous proteins, and we have described the structure of one such de novo protein, 1b11, a segment-swapped tetramer with novel architecture. Here, we have analyzed the thermodynamic stability and folding kinetics of the 1b11 tetramer and its monomeric and dimeric intermediates, and of 1b11 mutants with changes at the domain interface. Denatured 1b11 polypeptides fold into transient, folded monomers with marginal stability (DeltaG<1kcalmol(-1)) which convert rapidly ( approximately 6x10(4)M(-1)s(-1)) into dimers (DeltaG=9.8kcal/mol) and then more slowly ( approximately 3M(-1)s(-1)) into tetramers (DeltaG=28kcalmol(-1)). Segment swapping takes place during dimerization, as suggested by mass spectroscopic analysis of covalently linked peptides derived from proteolysis of a disulfide-linked dimer. Our results confirm that segment swapping and associated oligomerization are both powerful ways of stabilizing proteins, and we suggest that this may have been a feature of early protein evolution.