Refining a mouse model of progressive supranuclear palsy through inoculation of human post-mortem brain-derived tau.

Neuropathology of progressive supranuclear palsy after treatment with tilavonemab

Accelerated Human Mutant Tau Aggregation by Knocking Out Murine Tau in a Transgenic Mouse Model

The in vitro phosphorylation of the microtubule-associated protein tau by casein kinase II was studied. Purified human brain tau was phosphorylated by casein kinase II to a stoichiometry of 0.7 mol of 32P/mol of tau. Individual recombinant human tau isoforms were phosphorylated to stoichiometries ranging from 0.2 to 0.8 mol of 32P/mol of tau. Casein kinase II catalyzed a 4-fold greater incorporation of phosphate into the tau isoform containing a 58-amino acid insert near its amino terminus (T4L) than the isoforms without the 58-amino acid insert (T3 and T4). Phosphopeptide mapping of casein kinase II phosphorylated human tau and recombinant tau isoforms suggested that the isoforms containing an amino-terminal insert constitute the major substrates for casein kinase II within the tau family. The sites of phosphorylation on T4L were identified by digesting phosphorylated T4L with the protease Asp-N, separating the peptides by reversed phase high performance liquid chromatography, and analyzing the isolated peptides by liquid-secondary ion mass spectrometry and solid-phase amino-terminal sequencing. Thr39 was identified as the predominant phosphorylation site, which is located 5 residues from the amino-terminal insert in T4L. Phosphopeptide mapping of tau isolated from LA-N-5 neuroblastoma cells indicates that Thr39 is phosphorylated in situ. To our knowledge, this is the first demonstration of a differential phosphorylation of the human tau isoforms, with the isoforms containing the acidic amino-terminal insert being the preferred substrates of casein kinase II.

Does the Anti-Tau Strategy in Progressive Supranuclear Palsy Need to Be Reconsidered? No.

The most common neurodegenerative diseases are characterized by the presence of abnormal filamentous protein inclusions in nerve cells of the brain. In Alzheimer's disease, these inclusions are made of hyperphosphorylated tau protein (1). Together with the extracellular β-amyloid deposits, they constitute the defining neuropathological characteristics of Alzheimer's disease. Tau inclusions, in the absence of extracellular deposits, are characteristic of progressive supranuclear palsy, corticobasal degeneration, Pick's disease, argyrophilic grain disease, and frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) (1). The identification of mutations in Tau in FTDP-17 (2–4) has established that dysfunction of tau protein is central to the neurodegenerative process. At an experimental level, the expression of mutant human tau in nerve cells is leading to improved models of neurodegeneration. In this issue of PNAS, Kraemer et al. (5) describe lines of Caenorhabditis elegans expressing transgenic wild-type and mutant human tau protein. They represent an important addition to existing transgenic models for the human tauopathies. Tau protein is widely expressed in the mammalian nervous system, where it plays a role in the assembly and stabilization of microtubules (1). In adult human brain, there are six isoforms of tau, produced from a single gene by alternative mRNA splicing (6, 7). They differ by the presence or absence of a 29- or 58-aa insert in the N-terminal half and by the inclusion, or not, of a 31-aa repeat, encoded by exon 10 of Tau, in the C-terminal half of the protein (Fig. 1a). The exclusion of exon 10 leads to the production of three isoforms, each containing three repeats, and its inclusion leads to a further three isoforms, each containing four repeats. The repeats constitute the microtubule-binding region of tau, and similar levels of Three- And four-repeat isoforms are expressed in adult human brain. Repeat sequences homologous to those in tau are also present in the high-molecular-weight microtubule-associated proteins MAP2 and MAP4. The genomes of C. elegans and Drosophila melanogaster encode only one protein with tau-like repeats. Fig. 1. Mutations in Tau in FTDP-17. (a) Schematic diagram of the six tau isoforms (352–441 aa) expressed in adult human brain, with mutations in the coding region indicated by using the numbering of the 441-aa isoform. The six isoforms are produced ... Tau mutations in FTDP-17 are either missense, deletion, or silent mutations in the coding region, or intronic mutations located close to the splice-donor site of the intron following exon 10 (1). So far, 31 different mutations have been described in >80 families with FTDP-17 (Fig. 1). Functionally, Tau mutations fall into two largely nonoverlapping categories, those that influence the alternative splicing of tau pre-mRNA and those whose primary effect is at the protein level. The intronic mutations and most coding region mutations in exon 10 increase the splicing of this exon, changing the ratio between three- and four-repeat isoforms (3, 4). Approximately half of the known mutations have their primary effect at the RNA level. They affect exon splicing enhancer and silencer sequences in exon 10 (8) or destabilize a predicted stem-loop structure located at the boundary between exon 10 and the intron that follows it (3, 4, 9) (Fig. 1b). Thus, to a significant extent, FTDP-17 is a disease of alternative mRNA splicing. The other mutations affect tau isoforms directly. In accordance with their location in the microtubule-binding region, most missense mutations and the deletion mutations lead to a reduced ability of tau to promote microtubule assembly (10, 11). A number of mutations may cause FTDP-17, at least in part, by promoting the assembly of tau into filaments (12, 13). Kraemer et al. (5) expressed the 412-aa isoform of human tau in nerve cells of C. elegans, either in the wild-type form or with a missense mutation of FTDP-17 (P301L or V337M, Fig. 1a). This resulted in a reduced lifespan, behavioral impairment, defective cholinergic neurotransmission, the accumulation of insoluble phosphorylated tau, and neurodegeneration, as indicated by axonal damage and nerve cell loss. Although similar changes were observed in all transgenic lines, expression of mutant tau resulted in a more severe neurodegenerative phenotype than expression of wild-type tau. Soluble, phosphorylated tau was expressed 1 day after hatching, when the nervous system looked normal. However, uncoordinated behavior was already apparent in some lines, indicating that the simple expression of mutant human tau was sufficient to cause nerve cell dysfunction. By immunohistochemistry, tau staining was observed in most nerve cells and their processes. Insoluble, phosphorylated tau was first detected 5–7 days after hatching. Its appearance was paralleled by progressive axonal degeneration and nerve cell loss, which were more severe in mutant than in wild-type tau lines. By electron microscopy, 9-day-old worms from the mutant lines showed degenerating axons throughout the ventral and dorsal nerve cords. By immunoelectron microscopy, tau staining in the axons was largely diffuse. Amorphous, tau-positive aggregates were only observed in worms transgenic for V337M tau. How do these molecular and cellular changes compare with what has been observed in human diseases with tau pathology? Tauopathies in worms and humans share the progressive accumulation of insoluble tau and extensive neurodegeneration. However, there also some differences, the most conspicuous of which are a more modest phosphorylation and the absence of tau filaments in C. elegans. This is reminiscent of work in D. melanogaster, where the expression of wild-type and mutant human tau proteins in nerve cells led to a reduced lifespan and the loss of nerve cells, in the absence of tau filaments (14, 15). Phosphorylation of tau appeared to be more extensive in the fly than in the worm. Moreover, coexpression of human tau with the fly homologue of glycogen synthase kinase-3β, a candidate protein kinase for the hyperphosphorylation of tau, resulted in accelerated neurodegeneration and the formation of tau-immunoreactive inclusions (15). Filamentous structures were observed, but they were not shown to be decorated by anti-tau antibodies. In contrast to what has been described in FTDP-17 (16), tau-induced neurodegeneration involved programmed cell death (15). Taken together, it appears that conformationally altered, nonfilamentous human tau protein is neurotoxic in invertebrates. Tauopathies in worms and humans share the accumulation of insoluble tau and extensive neurodegeneration. What about vertebrate models for the human tauopathies? Expression of wild-type human tau in nerve cells of the sea lamprey has been shown to lead to the accumulation of filaments made of hyperphosphorylated tau and degenerative changes (17, 18), indicating a possible link between the formation of tau filaments and the degeneration of nerve cells. Most work has been done in the mouse (19–30), where the transgenic expression of single isoforms of wild-type human tau resulted in large numbers of nerve cells with abnormal tau-immunoreactive cell bodies and dendrites, as well as signs of axonal degeneration and muscle weakness (19–23). Abundant tau filaments were not observed, neither was substantial nerve cell loss. The level of tau phosphorylation was lower than in the human tauopathies. Overexpression of nonfilamentous tau can thus cause axonopathy and muscle weakness in the mouse. However, these transgenic lines are incomplete models for the human tauopathies. Mouse lines expressing single tau isoforms with missense mutations of FTDP-17 in nerve cells have provided more complete models (25–30). Some of these lines exhibited the essential molecular and cellular features of human tauopathies, including the formation of abundant filaments made of hyperphosphorylated tau protein and nonapoptotic nerve cell loss (25, 29). Filamentous tau was hyperphosphorylated to the same extent as in human tauopathy brains, and most of the same sites were also phosphorylated in soluble tau, consistent with the view that hyperphosphorylation precedes filament assembly. In a recent study, an increase in the phosphorylation of soluble tau resulted in increased filament formation, suggesting that phosphorylation of tau can drive filament formation (31). Work on mouse models has shown a correlation between the formation of tau filaments and neurodegeneration, but it is not known whether the two phenomena are causally related. The findings in C. elegans and D. melanogaster suggest that this may not be the case, because neurodegeneration was effected by nonfilamentous tau of reduced solubility. Alternatively, the mechanisms of neurodegeneration in worms and flies may differ from those in mice and humans. Disease models in C. elegans offer some advantages over mouse models, in particular with regard to the speed and relative ease with which genetic modifiers of disease phenotype can be discovered and pharmacological modifiers can be screened. Once identified, their relevance for mammalian model systems can be tested. In the future, this combined approach is likely to lead to a better understanding of the detailed molecular mechanisms by which the dysfunction of tau protein can cause neurodegeneration.

Progressive supranuclear palsy is a primary tauopathy affecting both neurons and glia and is responsible for both motor and cognitive symptoms. Recently, it has been suggested that progressive supranuclear palsy tauopathy may spread in the brain from cell to cell in a 'prion-like' manner. However, direct experimental evidence of this phenomenon, and its consequences on brain functions, is still lacking in primates. In this study, we first derived sarkosyl-insoluble tau fractions from post-mortem brains of patients with progressive supranuclear palsy. We also isolated the same fraction from age-matched control brains. Compared to control extracts, the in vitro characterization of progressive supranuclear palsy-tau fractions demonstrated a high seeding activity in P301S-tau expressing cells, displaying after incubation abnormally phosphorylated (AT8- and AT100-positivity), misfolded, filamentous (pentameric formyl thiophene acetic acid positive) and sarkosyl-insoluble tau. We bilaterally injected two male rhesus macaques in the supranigral area with this fraction of progressive supranuclear palsy-tau proteopathic seeds, and two other macaques with the control fraction. The quantitative analysis of kinematic features revealed that progressive supranuclear palsy-tau injected macaques exhibited symptoms suggestive of parkinsonism as early as 6 months after injection, remaining present until euthanasia at 18 months. An object retrieval task showed the progressive appearance of a significant dysexecutive syndrome in progressive supranuclear palsy-tau injected macaques compared to controls. We found AT8-positive staining and 4R-tau inclusions only in progressive supranuclear palsy-tau injected macaques. Characteristic pathological hallmarks of progressive supranuclear palsy, including globose and neurofibrillary tangles, tufted astrocytes and coiled bodies, were found close to the injection sites but also in connected brain regions that are known to be affected in progressive supranuclear palsy (striatum, pallidum, thalamus). Interestingly, while glial AT8-positive lesions were the most frequent near the injection site, we found mainly neuronal inclusions in the remote brain area, consistent with a neuronal transsynaptic spreading of the disease. Our results demonstrate that progressive supranuclear palsy patient-derived tau aggregates can induce motor and behavioural impairments in non-human primates related to the prion-like seeding and spreading of typical pathological progressive supranuclear palsy lesions. This pilot study paves the way for supporting progressive supranuclear palsy-tau injected macaque as a relevant animal model to accelerate drug development targeting this rare and fatal neurodegenerative disease.

BackgroundPSP is characterized by 4‐repeat tau accumulation in neuronal neurofibrillary tangles, oligodendroglial coiled bodies and in tufted astrocytes (a pathognomonic feature). In PSP, misfolded tau seeds the aggregation of tau throughout the brain in a prion‐like manner, with each cytopathology having a distinct, and sometimes overlapping pattern of distribution. The contribution of each of these three distinct cytopathologies to the accumulation and propagation of pathogenic tau throughout the PSP brain is not fully understood. To study this, we will develop a primary culture model of tau cytopathologies in PSP.MethodProgenitor cells were isolated from P2‐P4 6hTau mice, that express all six human tau isoforms. Cells were cultured in progenitor media and differentiated into astrocytes and neurons using media specific for each cell type and treated with human PSP and Alzheimer’s’ tau.ResultAstrocytes and neurons were labeled with GFAP and MAP2, respectively, to confirm their identity and purity. We will next introduce human brain‐derived PSP tau seeds into our cultures to investigate tau uptake and propagation. Initial studies will identify the optimal tau dose for aggregation and potency of tau in these cells. Then, using an astrocyte‐neuron co‐culture model we will assess how each cell type contributes to tau spread. Finally, we will explore the effects of tau accumulation on neurodegeneration.ConclusionThis study presents work towards developing an innovative cell culture model to investigate the role of different cytopathologies in the pathogenesis of PSP. The use of cell cultures enables the future incorporation of live imaging of tau trafficking between astrocytes and neurons. This model aims to deepen our understanding of PSP pathogenesis and provide a valuable platform for testing potential therapeutic strategies.

Tau aggregates are present in multiple neurodegenerative diseases known as “tauopathies,” including Alzheimer’s disease, Pick’s disease, progressive supranuclear palsy, and corticobasal degeneration. Such misfolded tau aggregates are therefore potential sources for selective detection and biomarker discovery. Six human tau isoforms present in brain tissues and both 3R and 4R isoforms have been observed in the neuronal inclusions. To develop selective markers for AD and related rare tauopathies, we first used an engineered tau protein fragment 4RCF as the substrate for ultrasensitive real-time quaking-induced conversion analyses (RT-QuIC). We showed that misfolded tau from diseased AD and other tauopathy brains were able to seed recombinant 4RCF substrate. We further expanded to use six individual recombinant tau isoforms as substrates to amplify misfolded tau seeds from AD brains. We demonstrated, for the first time to our knowledge, that misfolded tau from the postmortem AD brain tissues was able to specifically seed all six full-length human tau isoforms. Our results demonstrated that RT-QuIC analysis can discriminate AD and other tauopathies from non-AD normal controls. We further uncovered that 3R-tau isoforms displayed significantly faster aggregation kinetics than their 4R-tau counterparts under conditions of both no seeding and seeding with AD brain homogenates. In summary, our work offers potential new avenues of misfolded tau detection as potential biomarkers for diagnosis of AD and related tauopathies and provides new insights into isoform-specific human tau aggregation.

Alzheimer's Disease-Like Tau Neuropathology Leads to Memory Deficits and Loss of Functional Synapses in a Novel Mutated Tau Transgenic Mouse without Any Motor Deficits

We report on a novel transgenic mouse model expressing human full-length Tau with the Tau mutation A152T (hTau(AT)), a risk factor for FTD-spectrum disorders including PSP and CBD Brain neurons reveal pathological Tau conformation, hyperphosphorylation, mis-sorting, aggregation, neuronal degeneration, and progressive loss, most prominently in area CA3 of the hippocampus. The mossy fiber pathway shows enhanced basal synaptic transmission without changes in short- or long-term plasticity. In organotypic hippocampal slices, extracellular glutamate increases early above control levels, followed by a rise in neurotoxicity. These changes are normalized by inhibiting neurotransmitter release or by blocking voltage-gated sodium channels. CA3 neurons show elevated intracellular calcium during rest and after activity induction which is sensitive to NR2B antagonizing drugs, demonstrating a pivotal role of extrasynaptic NMDA receptors. Slices show pronounced epileptiform activity and axonal sprouting of mossy fibers. Excitotoxic neuronal death is ameliorated by ceftriaxone, which stimulates astrocytic glutamate uptake via the transporter EAAT2/GLT1. In summary, hTau(AT) causes excitotoxicity mediated by NR2B-containing NMDA receptors due to enhanced extracellular glutamate.

In the adult human brain, six isoforms of the microtubule-associated protein TAU are expressed, which result from alternative splicing of exons 2, 3, and 10 of the MAPT gene. These isoforms differ in the number of N-terminal inserts (0N, 1N, 2N) and C-terminal repeat domains (3R or 4R) and are differentially expressed depending on the brain region and developmental stage. Although all TAU isoforms can aggregate and form neurofibrillary tangles, some tauopathies, such as Pick’s disease and progressive supranuclear palsy, are characterized by the accumulation of specific TAU isoforms. The influence of the individual TAU isoforms in a cellular context, however, is understudied. In this report, we investigated the subcellular localization of the human-specific TAU isoforms in primary mouse neurons and analyzed TAU isoform-specific effects on cell area and microtubule dynamics in human SH-SY5Y neuroblastoma cells. Our results show that 2N-TAU isoforms are particularly retained from axonal sorting and that axonal enrichment is independent of the number of repeat domains, but that the additional repeat domain of 4R-TAU isoforms results in a general reduction of cell size and an increase of microtubule counts in cells expressing these specific isoforms. Our study points out that individual TAU isoforms may influence microtubule dynamics differentially both by different sorting patterns and by direct effects on microtubule dynamics.

Tau is normally a highly soluble phosphoprotein found predominantly in neurons. Six different isoforms of tau are expressed in the adult human CNS. Under pathological conditions, phosphorylated tau aggregates are a defining feature of neurodegenerative disorders called tauopathies. Recent findings have suggested a potential role of the gut-brain axis in CNS homeostasis, and therefore we set out to examine the isoform profile and phosphorylation state of tau in the enteric nervous system (ENS) under physiological conditions and in tauopathies. Surgical specimens of human colon from controls, Parkinson’s disease (PD) and progressive supranuclear palsy (PSP) patients were analyzed by Western Blot and immunohistochemistry using a panel of anti-tau antibodies. We found that adult human ENS primarily expresses two tau isoforms, localized in the cell bodies and neuronal processes. We did not observe any difference in the enteric tau isoform profile and phosphorylation state between PSP, PD and control subjects. The htau mouse model of tauopathy also expressed two main isoforms of human tau in the ENS, and there were no apparent differences in ENS tau localization or phosphorylation between wild-type and htau mice. Tau in both human and mouse ENS was found to be phosphorylated but poorly susceptible to dephosphorylation with lambda phosphatase. To investigate ENS tau phosphorylation further, primary cultures from rat enteric neurons, which express four isoforms of tau, were pharmacologically manipulated to show that ENS tau phosphorylation state can be regulated, at least in vitro. Our study is the first to characterize tau in the rodent and human ENS. As a whole, our findings provide a basis to unravel the functions of tau in the ENS and to further investigate the possibility of pathological changes in enteric neuropathies and tauopathies.

Accumulation of highly post-translationally modified tau proteins is a hallmark of neurodegenerative disorders known as tauopathies, the most common of which is Alzheimer's disease. Although six tau isoforms are found in the human brain, the majority of animal and cellular tauopathy models utilize a representative single isoform. However, the six human tau isoforms present overlapping but distinct distributions in the brain and are differentially involved in particular tauopathies. These observations support the notion that tau isoforms possess distinct functional properties important for both physiology and pathology. To address this hypothesis, the six human brain tau isoforms were expressed singly in the Drosophila brain and their effects in an established battery of assays measuring neuronal dysfunction, vulnerability to oxidative stress and life span were systematically assessed comparatively. The results reveal isoform-specific effects clearly not attributed to differences in expression levels but correlated with the number of microtubule-binding repeats and the accumulation of a particular isoform in support of the functional differentiation of these tau isoforms. Delineation of isoform-specific effects is essential to understand the apparent differential involvement of each tau isoform in tauopathies and their contribution to neuronal dysfunction and toxicity.

The microtubule-associated protein tau aggregates intracellularly by unknown mechanisms in Alzheimer's disease and other tauopathies. A contributing factor may be a failure to break down free cytosolic tau, thus creating a surplus for aggregation, although the proteases that degrade tau in brain remain unknown. To address this issue, we prepared cytosolic fractions from five normal human brains and from perfused rat brains and incubated them with or without protease inhibitors. D-Phenylalanyl-L-prolylarginyl chloromethyl ketone, a thrombin-specific inhibitor, prevented tau breakdown in these fractions, suggesting that thrombin is a brain protease that processes tau. We next exposed human recombinant tau to purified human thrombin and analyzed the fragments by N-terminal sequencing. We found that thrombin proteolyzed tau at multiple arginine and lysine sites. These include Arg(155)-Gly(156), Arg(209)-Ser(210), Arg(230)-Thr(231), Lys(257)-Ser(258), and Lys(340)-Ser(341) (numbering according to the longest human tau isoform). Temporally, the initial cleavage occurred at the Arg(155)-Gly(156) bond. Proteolysis of the resultant C-terminal tau fragment then proceeded bidirectionally. When tau was phosphorylated by glycogen synthase kinase-3beta, most of these proteolytic processes were inhibited, except for the first cleavage at the Arg(155)-Gly(156) bond. Furthermore, paired helical filament tau prepared from Alzheimer's disease brain was more resistant to thrombin proteolysis than following dephosphorylation by alkaline phosphatase. The results suggest a possible role for thrombin in proteolysis of tau under physiological and/or pathological conditions in human brains. They are consistent with the hypothesis that phosphorylation of tau inhibits proteolysis by thrombin or other endogenous proteases, leading to aggregation of tau into insoluble fibrils.

Classification of diseases with accumulation of Tau protein.