4R Tau Research Articles

Investigating astrocytes as mediators of tau spread

AbstractBackgroundHighly phosphorylated tau aggregates are believed to spread in a prion‐like manner through tauopathy brain. We set out to determine the characteristics of tau deposition in mice expressing wild‐type human 3R and 4R tau, following peripheral injection of tau extracts from different tauopathies. Tau accumulations identified around astrocytes in the experimental mice prompted us to develop a human astrocyte culture model to further investigate tau uptake and spread.MethodTau was isolated from human postmortem control (CTRL), Alzheimer’s disease (AD), progressive supranuclear palsy (PSP) and Pick’s disease (PiD) brain. Extracts were injected intraperitoneally into 4‐month old wild‐type and htau mice, and at 18 month‐of‐age tissue was collected for biochemical and immunohistochemical assessment.Human iPSCs were directed into neural progenitor cells and terminally plated as astrocytes using a previously established protocol. Astrocytes were characterised using RT‐qPCR and immunocytochemical assessment. Cultures were spiked with the previously derived pathogenic tau isolated from human tissue. Cells were analysed immunocytochemically for tau uptake.ResultWe found that peripheral injection of brain extracts from AD, PSP and PiD brain results in regional differences in the patterns of tau aggregate accumulation in the mouse brain. Some tau pathology was associated with ‘activated’ astrocytes, similar to the pathology observed in the corresponding human postmortem brain. Cultures of human astrocytes that express mature cell markers were established.ConclusionOur data suggests that neurons in different regions are susceptible to different species of tau aggregates. It is possible that this might hint towards selective vulnerability in different tauopathies. The association of tau pathology with astrocytes in some cases suggests that astrocytes may play a role in tau spread. Our human astrocyte cultures will provide a model to determine how astrocytes may facilitate the spread of tau and potentially contribute to selective regional vulnerability in tauopathies.

Early tau phosphorylation as a potential retinal biomarker for AD and other tauopathies

AbstractBackgroundThere is increased interest in in vivo labeling of Alzheimer’s disease (AD) pathology in the retina as a non‐invasive diagnostic approach (Jiang, Wang, Li, Cao, & Li, 2016; Koronyo et al., 2017). While the majority of studies focus on the detection of amyloid beta (Aβ) in the retina, the characterization of tau related pathology is scarce. In this study we assessed the presence of different tau isoforms in the retina in AD as well as other neurodegenerative diseases associated with and without tau pathology.MethodPost mortem eyes were collected through the Netherlands Brain Bank from donors with AD (n=6), progressive supranuclear palsy (PSP, n=2), frontotemporal lobar degeneration (FTLD; FUS and TDP43) (n=4), Lewy body disease (LBD, n=2), multiple system atrophy (MSA, n=2), Parkinson’s disease (PD, n=6), and non‐neurodegenerative controls (n=10). Superior‐temporal quadrants were cut in 10 µm sections. Sections were immunostained using primary antibodies directed against total tau (HT7), early tau phosphorylation (AT8), 3R and 4R tau isoforms (RD3/RD4) and late tau phosphorylation (pS422). The frontal cortex was used as a positive control.ResultTotal tau (HT7) was observed in non‐demented control and neurodegenerative cases (Figure 1). The presence of 3‐ and 4‐repeat isoforms of tau varied within the inner plexiform layer (IPL) and outer plexiform layer (OPL) with more 3‐repeat tau in the IPL and more 4‐repeat tau in the OPL. Only AD and PSP cases showed positive immunoreactivity for AT8, which was found differently distributed in the IPL and OPL (Figure 2). Tau phosphorylated at Ser422 was negative in all groups.ConclusionIn control and neurodegenerative cases, high levels of tau are present in the retina, mainly in the plexiform layers. Interestingly, we found different intensities for 3‐ and 4‐repeat tau in the IPL and OPL, implicating differences in tau expression by amacrine and horizontal cells (Chidlow, Wood, Manavis, Finnie, & Casson, 2017). Tau phosphorylated at Ser202/Thr205 clearly differentiates tauopathies from other neurodegenerative disease and non‐demented controls. Depending on the epitope, phosphorylated tau is a potential retinal biomarker for AD and other tauopathies.

CLK inhibition alters tau splicing and has a neuroprotective effect in vitro

AbstractBackgroundMicrotubule‐associated protein tau (MAPT) undergoes alternative splicing at exon 10 which encodes one of its four microtubule‐binding repeats. Exon 10 inclusion gives rise to tau isoforms with all four repeats (4R) whilst exon 10 exclusion leads to isoforms with three repeats (3R). The 1:1 ratio between 3R and 4R isoforms is tightly regulated by splicing machinery and crucial to normal function. Kinases such as CLKs regulate the activity of splicing factors and their interaction with target transcripts, including MAPT. Disruption to this balance leads to the formation of neurofibrillary tangles (NFTs) and neurodegeneration but the mechanism of this is not yet wholly understood.MethodsDifferentiated Neuro2a and SH‐SY5Y cells were used as neuronal models as these cell lines show an imbalance of MAPT isoforms with bias towards 4R and 3R respectively. Splicing factor SRSF1 was transiently knocked down to confirm its role in MAPT splicing. Novel kinase inhibitors were used to reduce the activity of CLKs and determine the effect on MAPT splicing. The expression ratio of 4R and 3R isoforms was measured by RT‐PCR. Okadaic acid (OA) was used to produce a neurotoxic model and investigate the use of kinase inhibitors for neuronal protection. As a functional measure β3‐tubulin fluorescent marker was used to quantify OA dependent reduction in neurite outgrowth.Results4R:3R tau expression significantly decreased by 65% in Neuro2a cells with knockdown of SRSF1 (students t‐test, p<0.001). CLK inhibition increased 4R:3R by 50% in SH‐SY5Y cells compared to control (one‐way ANOVA, p<0.05). SHSY5Y cells neurite outgrowth was significantly reduced by 60% and 85% with 1nM and 5nM OA treatment respectively, demonstrating a concentration dependent effect (one‐way ANOVA, p<0.001). At 1nM OA, neurite outgrowth was rescued by co‐treatment with a novel CLK inhibitor (two‐way ANOVA, p<0.0001).ConclusionsNovel CLK inhibitors can modulate the expression of MAPT splicing isoforms and exert a functional effect on neurite outgrowth. This suggests these compounds could have a therapeutic effect by rectifying the balance of 4R:3R tau expression.

Absolute quantification of tau isoforms in human brain by mass spectrometry

AbstractBackgroundTauopathies are neurodegenerative disorders characterized by the brain deposition of tau protein. Characterizing the biochemical alterations of tau in vivo is challenging given its low concentration in human fluids. Immunoassays can detect tau in the cerebrospinal fluid (CSF), but cannot provide a thorough characterization (isoforms, post‐translational modifications (Fig1). Mass Spectrometry (MS) allows in vivo detection with high sensitivity and absolute quantification of any biochemical characteristics of interest. We aim to characterize tau in the CSF using MS in patients using both CSF and brain samples available. We present here preliminary results on the first brain sample analysed.MethodFrozen brain samples of an AD patient (Braak stage VI) were obtained from the UCLouvain donation centre. We prepared a neocortical homogenate (200 mg of wet weight/ml) that we ultra‐centrifugated to separate sarkosyl‐soluble tau from insoluble tau aggregates (Diner et al., 2017). Sarkosyl‐soluble tau was isolated by immunoprecipitation with 0.5 to 4 µg of mouse monoclonal HJ8.7 antibody. Immunoprecipitated tau was eluted with 0.1 % formic acid, dried and digested with 250ng trypsin. Liquid Chromatography (LC)‐MS/MS analysis was performed using parallel reaction monitoring (PRM) to identify tau isoforms. Specific peptides for each isoform were targeted to ensure selective and sensitive detection. In parallel, we diluted this extract 5000 times (2µl in 10 ml) and harvested 1 ml of sample into which we spiked 300pg of recombinant isotopically labelled full‐length (2N‐4R) tau protein (15N labelling). This allowed us to detect 15N isotopologues of 2N and 4R isoforms and quantify endogenous tau via intensity ratios.ResultWe detected all tau isoforms (0N, 1N, 2N, 3R, 4R) by post‐acquisition analysis of the data with Skyline (Fig.2). We absolutely quantified isoforms 2N and 4R separately from other isoforms in our sarkosyl‐soluble protein extract using 15N‐tau and obtained 150 pg/µl of tau 2N and 390 pg/µl of tau 4R (Fig.3).ConclusionThis targeted MS analysis proved to be effective for detecting and quantifying tau isoforms in the brain of one AD patient. We will soon compare this result with CSF coming from the same patient, and test CSF and brain samples coming from patients presenting non‐AD tauopathies.

Tau accumulation in astrocytes of the dentate gyrus induces neuronal dysfunction and memory deficits in Alzheimer's disease.

Alzheimer's disease (AD) is characterized by the accumulation of the tau protein in neurons, neurodegeneration and memory loss. However, the role of non-neuronal cells in this chain of events remains unclear. In the present study, we found accumulation of tau in hilar astrocytes of the dentate gyrus of individuals with AD. In mice, the overexpression of 3R tau specifically in hilar astrocytes of the dentate gyrus altered mitochondrial dynamics and function. In turn, these changes led to a reduction of adult neurogenesis, parvalbumin-expressing neurons, inhibitory synapses and hilar gamma oscillations, which were accompanied by impaired spatial memory performances. Together, these results indicate that the loss of tau homeostasis in hilar astrocytes of the dentate gyrus is sufficient to induce AD-like symptoms, through the impairment of the neuronal network. These results are important for our understanding of disease mechanisms and underline the crucial role of astrocytes in hippocampal function.

Fibrillation and molecular characteristics are coherent with clinical and pathological features of 4-repeat tauopathy caused by MAPT variant G273R

Microtubule Associated Protein Tau (MAPT) forms proteopathic aggregates in several diseases. The G273R tau mutation, located in the first repeat region, was found by exome sequencing in a patient who presented with dementia and parkinsonism. We herein return to pathological examination which demonstrated tau immunoreactivity in neurons and glia consistent of mixed progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD) features. To rationalize the pathological findings, we used molecular biophysics to characterize the mutation in more detail in vitro and in Drosophila. The G273R mutation increases the aggregation propensity of 4-repeat (4R) tau and alters the tau binding affinity towards microtubules (MTs) and F-actin. Tau aggregates in PSP and CBD are predominantly 4R tau. Our data suggest that the G273R mutation induces a shift in pool of 4R tau by lower F-actin affinity, alters the conformation of MT bound 4R tau, while increasing chaperoning of 3R tau by binding stronger to F-actin. The mutation augmented fibrillation of 4R tau initiation in vitro and in glial cells in Drosophila and showed preferential seeding of 4R tau in vitro suggestively causing a late onset 4R tauopathy reminiscent of PSP and CBD.

The role of hypoxia on Alzheimer’s disease-related APP and Tau mRNA formation

The removal of introns from mRNA precursors (pre-mRNAs) is an essential step in eukaryotic gene expression. The splicing machinery heavily contributes to biological complexity and especially to the ability of cells to adapt to altered cellular conditions. Hypoxia also plays a key role in the pathophysiology of many diseases, including Alzheimer’s disease (AD). In the presented study, we have examined the influence of cellular hypoxia on mRNA splice variant formation from Alzheimer’s disease-related Tau and APP genes in brain cells. We have shown that the hypoxic microenvironment influenced the formation of Tau mRNA splice variants, but had no effect on APP mRNA splice variant formation. Additionally, our presented results indicate that splicing factor SRSF1 but not SRSF5 alters the formation of Tau cellular mRNA splice variants in hypoxic cells. Obtained results have also shown that hypoxic brain cells possess enhanced CLK1-4 kinase mRNA levels. This study underlines that cellular hypoxia can influence disease development through changing pre-mRNA splicing.

Neurofibrillary changes undergoing morphological and biochemical changes - How does tau with the profile shift of from four repeat to three repeat spread in Alzheimer brain?

The concept of the hierarchal spread of neurofibrillary tangles (NFTs) from the hippocampus to the cortex in Alzheimer's disease (AD)/aging brains, initially proposed by Braak and Braak, revolutionized our understanding by putatively explaining that tau lesions are being unidirectionally extended along neural connections. Because pathological misfolding of tau can serve as a seed to induce identical misfolding on normal tau, this prion-like property is considered to represent a molecular mechanism that may explain such lesion spread. However, double labeling studies for three repeat (3R) and four repeat (4R) tau demonstrated a profile shift in these tau isoforms along chronological change: initially positive only for 4R in early pretangles, gradual involvement of 3R in mature NFTs, and finally replaced by 3R in advanced ghost tangles. This profile shift is hardly explained by aggregation of a single tau molecule. Surprisingly, this profile shift from 4R to 3R tau is shared with the hierarchal spread of NFT around the hippocampus, which is hardly explained by a simple mechanism such as propagation of a single tau molecule. Some molecules other than tau or non-materialist influences such as neuronal activity may be candidate mechanisms to explain this regional profile shift if it is mediated by neural connections. Because this profile shift is shared with brainstem NFTs, it may instead represent a cell autonomous phenomenon, tightly linked with progression of AD but not necessarily linked with prion-like propagation along neural connections. In addition, we recently clarified that posttranslational deamidation of 4R tau at asparagine 279 to aspartate is a marker for AD, distinct from progressive supranuclear palsy (PSP)/corticobasal degeneration (CBD). This may provide another axis to see how AD-NFTs develop and how their 4R tau is distinct from 4R tau of PSP/CBD. Because tau species and their distributions are disease-specific, molecular homogenization, such as "tauopathy" to encompass AD, PSP/CBD and Pick body disease, is just an oversimplification that may obscure fundamental distinctions between human diseases. Careful reference to the realities of human brain disease may provide a solid and fruitful basis to improve diagnostic and therapeutic strategies to tackle these intractable diseases.

Hydration and Dynamics of Full-Length Tau Amyloid Fibrils Investigated by Solid-State Nuclear Magnetic Resonance.

The microtubule-associated protein tau aggregates into distinct neurofibrillary tangles in brains afflicted with multiple neurodegenerative diseases such as Alzheimer's disease and corticobasal degeneration (CBD). The mechanism of tau misfolding and aggregation is poorly understood. Determining the structure, dynamics, and water accessibility of tau filaments may provide insight into the pathway of tau misfolding. Here, we investigate the hydration and dynamics of the β-sheet core of heparin-fibrillized 0N4R tau using solid-state nuclear magnetic resonance spectroscopy. This β-sheet core consists of the second and third microtubule-binding repeats, R2 and R3, respectively, which form a hairpin. Water-edited two-dimensional (2D) 13C-13C and 15N-13C correlation spectra show that most residues in R2 and R3 domains have low water accessibility, indicating that this hairpin is surrounded by other proteinaceous segments. However, a small number of residues, especially S285 and S316, are well hydrated compared to other Ser and Thr residues, suggesting that there is a small water channel in the middle of the hairpin. To probe whether water accessibility correlates with protein dynamics, we measured the backbone N-H dipolar couplings of the β-sheet core. Interestingly, residues in the fourth microtubule-binding repeat, R4, show rigid-limit N-H dipolar couplings, even though this domain exhibits weaker intensities in the 2D 15N-13C correlation spectra. These results suggest that the R4 domain participates in cross-β hydrogen bonding in some of the subunits but exhibits dynamic disorder in other subunits. Taken together, these hydration and dynamics data indicate that the R2-R3 hairpin of 0N4R tau is shielded from water by other proteinaceous segments on the exterior but contains a small water pore in the interior. This structural topology has various similarities with the CBD tau fibril structure but also shows specific differences. The disorder of the R4 domain and the presence of a small water channel in the heparin-fibrillized 4R tau have implications for the structure of tau fibrils in diseased brains.

Tau immunophenotypes in chronic traumatic encephalopathy recapitulate those of ageing and Alzheimer's disease.

Traumatic brain injury (TBI) is a risk factor for neurodegenerative disease, including chronic traumatic encephalopathy (CTE). Preliminary consensus criteria define the pathognomonic lesion of CTE as patchy tau pathology within neurons and astrocytes at the depths of cortical sulci. However, the specific tau isoform composition and post-translational modifications in CTE remain largely unexplored. Using immunohistochemistry, we performed tau phenotyping of CTE neuropathologies and compared this to a range of tau pathologies, including Alzheimer's disease, primary age-related tauopathy, ageing-related tau astrogliopathy and multiple subtypes of frontotemporal lobar degeneration with tau inclusions. Cases satisfying preliminary consensus diagnostic criteria for CTE neuropathological change (CTE-NC) were identified (athletes, n = 10; long-term survivors of moderate or severe TBI, n = 4) from the Glasgow TBI Archive and Penn Neurodegenerative Disease Brain Bank. In addition, material from a range of autopsy-proven ageing-associated and primary tauopathies in which there was no known history of exposure to TBI was selected as non-injured controls (n = 32). Each case was then stained with a panel of tau antibodies specific for phospho-epitopes (PHF1, CP13, AT100, pS262), microtubule-binding repeat domains (3R, 4R), truncation (Tau-C3) or conformation (GT-7, GT-38) and the extent and distribution of staining assessed. Cell types were confirmed with double immunofluorescent labelling. Results demonstrate that astroglial tau pathology in CTE is composed of 4R-immunoreactive thorn-shaped astrocytes, echoing the morphology and immunophenotype of astrocytes encountered in ageing-related tau astrogliopathy. In contrast, neurofibrillary tangles of CTE contain both 3R and 4R tau, with post-translational modifications and conformations consistent with Alzheimer's disease and primary age-related tauopathy. Our observations establish that the astroglial and neurofibrillary tau pathologies of CTE are phenotypically distinct from each other and recapitulate the tau immunophenotypes encountered in ageing and Alzheimer's disease. As such, the immunohistochemical distinction of CTE neuropathology from other mixed 3R/4R tauopathies of Alzheimer's disease and ageing may rest solely on the pattern and distribution of pathology.

Increasing Tau 4R Tau Levels Exacerbates Hippocampal Tau Hyperphosphorylation in the hTau Model of Tauopathy but Also Tau Dephosphorylation Following Acute Systemic Inflammation.

Inflammation is considered a mechanistic driver of Alzheimer's disease, thought to increase tau phosphorylation, the first step to the formation of neurofibrillary tangles (NFTs). To further understand how inflammation impacts the development of tau pathology, we used (hTau) mice, which express all six, non-mutated, human tau isoforms, but with an altered ratio of tau isoforms favoring 3R tau due to the concomitant loss of murine tau (mTau) that is predominantly 4R. Such an imbalance pattern has been related to susceptibility to NFTs formation, but whether or not this also affects susceptibility to systemic inflammation and related changes in tau phosphorylation is not known. To reduce the predominance of 3R tau by increasing 4R tau availability, we bred hTau mice on a heterozygous mTau background and compared the impact of systemic inflammation induced by lipopolysaccharide (LPS) in hTau mice hetero- or homozygous mTau knockout. Three-month-old male wild-type (Wt), mTau+/−, mTau−/−, hTau/mTau+/−, and hTau/mTau−/− mice were administered 100, 250, or 330 μg/kg of LPS or its vehicle phosphate buffer saline (PBS) [intravenously (i.v.), n = 8–9/group]. Sickness behavior, reflected by behavioral suppression in the spontaneous alternation task, hippocampal tau phosphorylation, measured by western immunoblotting, and circulating cytokine levels were quantified 4 h after LPS administration. The persistence of the LPS effects (250 μg/kg) on these measures, and food burrowing behavior, was assessed at 24 h post-inoculation in Wt, mTau+/−, and hTau/mTau+/− mice (n = 9–10/group). In the absence of immune stimulation, increasing 4R tau levels in hTau/mTau+/− exacerbated pS202 and pS396/404 tau phosphorylation, without altering total tau levels or worsening early behavioral perturbations characteristic of hTau/mTau−/− mice. We also show for the first time that modulating 4R tau levels in hTau mice affects the response to systemic inflammation. Behavior was suppressed in all genotypes 4 h following LPS administration, but hTau/mTau+/− exhibited more severe sickness behavior at the 100 μg/kg dose and a milder behavioral and cytokine response than hTau/mTau−/− mice at the 330 μg/kg dose. All LPS doses decreased tau phosphorylation at both epitopes in hTau/mTau+/− mice, but pS202 levels were selectively reduced at the 100 μg/kg dose in hTau/mTau−/− mice. Behavioral suppression and decreased tau phosphorylation persisted at 24 h following LPS administration in hTau/mTau+/− mice.

A single ultrasensitive assay for detection and discrimination of tau aggregates of Alzheimer and Pick diseases

Multiple neurodegenerative diseases are characterized by aggregation of tau molecules. Adult humans express six isoforms of tau that contain either 3 or 4 microtubule binding repeats (3R or 4R tau). Different diseases involve preferential aggregation of 3R (e.g Pick disease), 4R (e.g. progressive supranuclear palsy), or both 3R and 4R tau molecules [e.g. Alzheimer disease and chronic traumatic encephalopathy]. Three ultrasensitive cell-free seed amplification assays [called tau real-time quaking induced conversion (tau RT-QuIC) assays] have been developed that preferentially detect 3R, 4R, or 3R/4R tau aggregates in biospecimens. In these reactions, low-fg amounts of a given self-propagating protein aggregate (the seed) are incubated with a vast excess of recombinant tau monomers (the substrate) in multi-well plates. Over time, the seeds incorporate the substrate to grow into amyloids that can then be detected using thioflavin T fluorescence. Here we describe a tau RT-QuIC assay (K12 RT-QuIC) that, using a C-terminally extended recombinant 3R tau substrate (K12CFh), enables sensitive detection of Pick disease, Alzheimer disease, and chronic traumatic encephalopathy seeds in brain homogenates. The discrimination of Pick disease from Alzheimer disease and chronic traumatic encephalopathy cases is then achieved through the quantitative differences in K12 RT-QuIC assay thioflavin T responses, which correlate with structural properties of the reaction products. In particular, Fourier transform infrared spectroscopy analysis of the respective K12CFh amyloids showed distinct β-sheet conformations, suggesting at least partial propagation of the original seed conformations in vitro. Thus, K12 RT-QuIC provides a single assay for ultrasensitive detection and discrimination of tau aggregates comprised mainly of 3R, or both 3R and 4R, tau isoforms.

Pre-clinical characterisation of E2814, a high-affinity antibody targeting the microtubule-binding repeat domain of tau for passive immunotherapy in Alzheimer\u2019s disease

Tau deposition in the brain is a pathological hallmark of many neurodegenerative disorders, including Alzheimer’s disease (AD). During the course of these tauopathies, tau spreads throughout the brain via synaptically-connected pathways. Such propagation of pathology is thought to be mediated by tau species (“seeds”) containing the microtubule binding region (MTBR) composed of either three repeat (3R) or four repeat (4R) isoforms. The tau MTBR also forms the core of the neuropathological filaments identified in AD brain and other tauopathies. Multiple approaches are being taken to limit tau pathology, including immunotherapy with anti-tau antibodies. Given its key structural role within fibrils, specifically targetting the MTBR with a therapeutic antibody to inhibit tau seeding and aggregation may be a promising strategy to provide disease-modifying treatment for AD and other tauopathies. Therefore, a monoclonal antibody generating campaign was initiated with focus on the MTBR. Herein we describe the pre-clinical generation and characterisation of E2814, a humanised, high affinity, IgG1 antibody recognising the tau MTBR. E2814 and its murine precursor, 7G6, as revealed by epitope mapping, are antibodies bi-epitopic for 4R and mono-epitopic for 3R tau isoforms because they bind to sequence motif HVPGG. Functionally, both antibodies inhibited tau aggregation in vitro. They also immunodepleted a variety of MTBR-containing tau protein species. In an in vivo model of tau seeding and transmission, attenuation of deposition of sarkosyl-insoluble tau in brain could also be observed in response to antibody treatment. In AD brain, E2814 bound different types of tau filaments as shown by immunogold labelling and recognised pathological tau structures by immunohistochemical staining. Tau fragments containing HVPGG epitopes were also found to be elevated in AD brain compared to PSP or control. Taken together, the data reported here have led to E2814 being proposed for clinical development.

Differences Between Human and Murine Tau at the N-terminal End.

Human tauopathies, such as Alzheimer’s disease (AD), have been widely studied in transgenic mice overexpressing human tau in the brain. The longest brain isoforms of Tau in mice and humans show 89% amino acid identity; however, the expression of the isoforms of this protein in the adult brain of the two species differs. Tau 3R isoforms are not present in adult mice. In contrast, the adult human brain contains Tau 3R and also Tau 4R isoforms. In addition, the N-terminal sequence of Tau protein in mice and humans differs, a Tau peptide (residues 17–28) being present in the latter but absent in the former. Here we review the main published data on this N-terminal sequence that suggests that human and mouse Tau proteins interact with different endogenous proteins and also show distinct secretion patterns.

Transmission of tauopathy strains is independent of their isoform composition

The deposition of pathological tau is a common feature in several neurodegenerative tauopathies. Although equal ratios of tau isoforms with 3 (3R) and 4 (4R) microtubule-binding repeats are expressed in the adult human brain, the pathological tau from different tauopathies have distinct isoform compositions and cell type specificities. The underlying mechanisms of tauopathies are unknown, partially due to the lack of proper models. Here, we generate a new transgenic mouse line expressing equal ratios of 3R and 4R human tau isoforms (6hTau mice). Intracerebral injections of distinct human tauopathy brain-derived tau strains into 6hTau mice recapitulate the deposition of pathological tau with distinct tau isoform compositions and cell type specificities as in human tauopathies. Moreover, through in vivo propagation of these tau strains among different mouse lines, we demonstrate that the transmission of distinct tau strains is independent of strain isoform compositions, but instead intrinsic to unique pathological conformations.

4-Repeat tau seeds and templating subtypes as brain and CSF biomarkers of frontotemporal lobar degeneration.

To address the need for more meaningful biomarkers of tauopathies, we have developed an ultrasensitive tau seed amplification assay (4R RT-QuIC) for the 4-repeat (4R) tau aggregates of progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and other diseases with 4R tauopathy. The assay detected seeds in 106-109-fold dilutions of 4R tauopathy brain tissue but was orders of magnitude less responsive to brain with other types of tauopathy, such as from Alzheimer's disease cases. The analytical sensitivity for synthetic 4R tau fibrils was ~ 50fM or 2fg/sample. A novel dimension of this tau RT-QuIC testing was the identification of three disease-associated classes of 4R tau seeds; these classes were revealed by conformational variations in the in vitro amplified tau fibrils as detected by thioflavin T fluorescence amplitudes and FTIR spectroscopy. Tau seeds were detected in postmortem cerebrospinal fluid (CSF) from all neuropathologically confirmed PSP and CBD cases but not in controls. CSF from living subjects had weaker seeding activities; however, mean assay responses for cases clinically diagnosed as PSP and CBD/corticobasal syndrome were significantly higher than those from control cases. Altogether, 4R RT-QuIC provides a practical cell-free method of detecting and subtyping pathologic 4R tau aggregates as biomarkers.

Tau isoform–specific stabilization of intermediate states during microtubule assembly and disassembly

The microtubule (MT)-associated protein tau regulates the critical growing and shortening behaviors of MTs, and its normal activity is essential for neuronal development and maintenance. Accordingly, aberrant tau action is tightly associated with Alzheimer's disease and is genetically linked to several additional neurodegenerative diseases known as tauopathies. Although tau is known to promote net MT growth and stability, the precise mechanistic details governing its regulation of MT dynamics remain unclear. Here, we have used the slowly-hydrolyzable GTP analog, guanylyl-(α,β)-methylene-diphosphonate (GMPCPP), to examine the structural effects of tau at MT ends that may otherwise be too transient to observe. The addition of both four-repeat (4R) and three-repeat (3R) tau isoforms to pre-formed GMPCPP MTs resulted in the formation of extended, multiprotofilament-wide projections at MT ends. Furthermore, at temperatures too low for assembly of bona fide MTs, both tau isoforms promoted the formation of long spiral ribbons from GMPCPP tubulin heterodimers. In addition, GMPCPP MTs undergoing cold-induced disassembly in the presence of 4R tau (and to a much lesser extent 3R tau) also formed spirals. Finally, three pathological tau mutations known to cause neurodegeneration and dementia were differentially compromised in their abilities to stabilize MT disassembly intermediates. Taken together, we propose that tau promotes the formation/stabilization of intermediate states in MT assembly and disassembly by promoting both longitudinal and lateral tubulin-tubulin contacts. We hypothesize that these activities represent fundamental aspects of tau action that normally occur at the GTP-rich ends of GTP/GDP MTs and that may be compromised in neurodegeneration-causing tau variants.

In vitro 0N4R tau fibrils contain a monomorphic β-sheet core enclosed by dynamically heterogeneous fuzzy coat segments

Misfolding of the microtubule-binding protein tau into filamentous aggregates is characteristic of many neurodegenerative diseases such as Alzheimer's disease and progressive supranuclear palsy. Determining the structures and dynamics of these tau fibrils is important for designing inhibitors against tau aggregation. Tau fibrils obtained from patient brains have been found by cryo-electron microscopy to adopt disease-specific molecular conformations. However, in vitro heparin-fibrillized 2N4R tau, which contains all four microtubule-binding repeats (4R), was recently found to adopt polymorphic structures. Here we use solid-state NMR spectroscopy to investigate the global fold and dynamics of heparin-fibrillized 0N4R tau. A single set of 13C and 15N chemical shifts was observed for residues in the four repeats, indicating a single β-sheet conformation for the fibril core. This rigid core spans the R2 and R3 repeats and adopts a hairpin-like fold that has similarities to but also clear differences from any of the polymorphic 2N4R folds. Obtaining a homogeneous fibril sample required careful purification of the protein and removal of any proteolytic fragments. A variety of experiments and polarization transfer from water and mobile side chains indicate that 0N4R tau fibrils exhibit heterogeneous dynamics: Outside the rigid R2-R3 core, the R1 and R4 repeats are semirigid even though they exhibit β-strand character and the proline-rich domains undergo large-amplitude anisotropic motions, whereas the two termini are nearly isotropically flexible. These results have significant implications for the structure and dynamics of 4R tau fibrils in vivo.

Multimodal neuroimaging relationships in progressive supranuclear palsy

Progressive supranuclear palsy is characterized primarily by 4R tau inclusions, atrophy in the brainstem and basal ganglia, and neurodegeneration along the dentatorubrothalamic tract, which are measurable in vivo using flortaucipir PET, T1-weighted MRI, and MRI with diffusion tensor imaging (DTI). However, little is known about how these processes relate to each other. The aim of this study was to investigate multimodal associations between flortaucipir PET uptake, tissue volume loss on structural MRI and white matter tract disruption on DTI. Thirty-four patients with progressive supranuclear palsy and 29 normal controls underwent flortaucipir PET, MRI and DTI. Voxel-wise comparison was performed between patients and controls. Sparse canonical correlations analysis was applied on regional measurements of flortaucipir uptake, tissue volume, fractional anisotropy and mean diffusivity of the PSP population. Pearson's correlation coefficients were assessed across modalities on the regions identified by the sparse canonical correlation analyses. Sparse canonical correlation analyses identified associations between elevated flortaucipir uptake in the cerebellar dentate, red nucleus and subthalamic nucleus and decreased volume in the same regions, and decreased fractional anisotropy and increased mean diffusivity in tracts including the superior cerebellar peduncle, sagittal striatum and posterior corona radiata. Furthermore, decreased fractional anisotropy and increased mean diffusivity in the body of the corpus callosum and anterior and superior corona radiata were related to volume loss in the frontal lobe. Tau uptake measured by flortaucipir PET appears to be related to the neurodegenerative process of progressive supranuclear palsy, including reduced tissue volume and white matter tract degeneration.

18F-PI2620 TAU-PET IN PROGRESSIVE SUPRANUCLEAR PALSY: A MULTI-CENTER EVALUATION

Progressive supranuclear palsy (PSP) is a 4-repeat (4R) tauopathy and region-specific tau deposits establish the neuropathological diagnosis of “definite PSP” post mortem. Future interventional trials against tau in PSP would strongly benefit from biomarkers to validate the specific presence of the target before therapy initiation. The novel second generation tau-PET ligand 18F-PI2620 proved absent off-target binding to monoamine oxidases and high affinity to 3/4R tau in Alzheimer's disease (AD). The aim of this multicenter-evaluation was to investigate 18F-PI2620 in patients with suspected 4R tau pathology in clinically diagnosed PSP. Eighteen patients (70±6y, n=9 female) with probable or possible PSP Richardson syndrome according to MDS-PSP criteria underwent 18F-PI2620 PET at four different centers together with ten healthy controls and ten disease controls (Multi-system atrophy, Parkinson's disease, and AD). Standardized uptake value ratios (SUVr, 30-60min) of predefined subcortical brain regions were generated using cerebellar scaling. SUVr data were compared between PSP, HC, and disease controls. Statistical parametric mapping (SPM, V12) was performed between PSP and HC. SUVr and SPM data were corrected for different centers. An in vitro pilot autoradiography using 18F-PI2620 incubation of brain slices was performed. Elevated 18F-PI2620 SUVr was observed in PSP patients (PSP rating scale: 40±17; range 13-71) in the globus pallidus (1.34±0.16; p=0.001; d=1.72) and the substantia nigra (1.33±0.13; p=0.002; d=1.52) when compared to HC (1.12±0.09/1.15±0.08). Disease controls showed a similar signal in the globus pallidus (1.12±0.08; p=n.s.) and a moderate elevation in the substantia nigra (1.25±0.10; p=n.s.) when compared to HC. SPM revealed elevated 18F-PI2620 uptake in the globus pallidus, in the substantia nigra, and frontal and parietal cortices (all p<0.001, uncorrected) as compared to HC. Subjects with low disease severity (PSP rating scale ≤30; n=4) already had a significantly elevated 18F-PI2620 uptake in the globus pallidus when compared to HC (1.38±0.13; p=0.001; d=2.32). Preliminary in vitro autoradiography showed distinguishable 18F-PI2620 binding in the globus pallidus of a PSP patient which was however lower when compared to cortical binding in AD.