4R Tau Research Articles

PROGRESS IN DEVELOPING A LIGHT‐STABLE 4R TAU PET IMAGING AGENT: APN‐1701 FIH

AbstractBackgroundThe need for a light stable 4R tau PET tracer for use in diverse tauopathies is widely appreciated. We aimed to assess the initial human imaging profile of [18F]APN‐1701 as such a tracer and to compare its imaging profile to the well‐established but light‐sensitive tracer [18F]‐APN‐1607 that can detect 4R aggregates as well as 3R and mixed 3R/4R aggregates. [18F]APN‐1607 has progressed to a multicenter Phase 2 clinical trial for Alzheimer’s disease (AD); therefore, prior imaging data with it was considered valuable for comparison.MethodOne cognitively normal (CN) subject (male, 73yo) and one AD subject (female, 58 yo), who had undergone prior amyloid profiling using [18F]florbetapir PET and subsequent imaging with [18F]APN‐1607 in a proof‐of‐concept study and a test‐retest study, respectively, participated in the current study. Dynamic imaging with [18F]APN‐1701 was performed over 180 min to obtain kinetic data.ResultOverall, for both subjects [18F]APN‐1701 generally matched [18F]APN‐1607 findings, i.e., peak SUV values reached similar levels. The CN subject, who was amyloid negative, had slight, uniform cortical [18F]‐APN‐1701 uptake and more substantial uptake in the pallidum, pons and midbrain. SUVr images for the AD subject showed retention in similar brain regions for both tau tracers with the same rank order of regional SUV/SUVr. Notably, for this subject [18F]APN‐1701 continued to accumulate up to the end of image acquisition.Conclusion[18F]APN‐1701 demonstrates cortical uptake generally similar to [18F]APN‐1607 but its slow kinetics are unfavorable for further development.

Subcortical tau‐accumulation predicts neuronal dysfunction in the cortex based on functional connectivity in 4R‐tauopathies

AbstractBackground4‐repeat (4R) tauopathies are neurodegenerative diseases characterized by cerebral accumulation of 4R tau pathology. The most prominent 4R‐tauopathies are progressive‐supranuclear‐palsy (PSP) and corticobasal‐syndrome (CBS) characterized by tau accumulation in subcortical nuclei as well as cortical neuronal dysfunction, as shown by PET‐assessed hypoperfusion and glucose hypometabolism. Yet, there is a spatial mismatch between subcortical tau deposition patterns and cortical neuronal dysfunction and it is unclear how these two pathological brain changes are interrelated. Here, we hypothesized that subcortical tau pathology induces diaschisis‐like neuronal dysfunction in functionally connected cortical regions.MethodWe included 47 patients with clinically diagnosed PSP or CBS who underwent structural MRI and 18F‐PI‐2620 tau‐PET. PI‐2620 PET was recorded using a dynamic one‐shot, two‐stop acquisition protocol, to determine an early 0.5‐2.5min post‐tracer‐injection perfusion window for assessing cortical neuroinjury in 200 cortical ROIs of the Schaefer atlas, as well as a 20‐40min post‐tracer‐injection window to determine 4R‐tau load in 32 subcortical ROIs of the TIAN atlas. We determined tau epicenters as 10% of subcortical ROIs with highest tau‐PET, and assessed the connectivity of tau epicenters to cortical ROIs using an age‐matched 3T resting‐state fMRI template derived from 69 healthy elderly. Using linear regression, we assessed whether i) higher subcortical tau‐PET was associated with overall reduced cortical perfusion and ii) whether cortical hypoperfusion was observed preferentially in regions closely connected to subcortical tau epicenters.ResultAs hypothesized, higher subcortical tau‐PET was associated with lower cortical perfusion (R=‐0,37, p‐value: <0,011, Fig.1). Using group‐average tau‐PET and perfusion‐PET, we found that the seed‐based connectivity pattern of subcortical tau epicenters predicted cortical perfusion patterns, where cortical regions that were more closely connected to the tau epicenter showed stronger hypoperfusion (R=‐0,16, p‐value: <0,023, Fig.2A). This association was also observed on the subject level, as indicated by overall negative b‐values of the association between tau epicenter connectivity and cortical perfusion (one‐sample t‐test: t‐value: ‐3,45, p‐value: <0,001, Fig.3).ConclusionIn 4R‐tauopathies subcortical tau‐accumulation is associated with remote neuronal dysfunction in functionally connected cortical regions. This suggests that subcortical tau pathology may induce diaschisis‐like cortical dysfunction, which may contribute to clinical disease manifestation and clinical heterogeneity.

PROGRESS IN DEVELOPING A LIGHT‐STABLE 4R TAU PET IMAGING AGENT: APN‐1701 FIH

AbstractBackgroundThe need for a light stable 4R tau PET tracer for use in diverse tauopathies is widely appreciated. We aimed to assess the initial human imaging profile of [18F]APN‐1701 as such a tracer and to compare its imaging profile to the well‐established but light‐sensitive tracer [18F]‐APN‐1607 that can detect 4R aggregates as well as 3R and mixed 3R/4R aggregates. [18F]APN‐1607 has progressed to a multicenter Phase 2 clinical trial for Alzheimer’s disease (AD); therefore, prior imaging data with it was considered valuable for comparison.MethodOne cognitively normal (CN) subject (male, 73yo) and one AD subject (female, 58 yo), who had undergone prior amyloid profiling using [18F]florbetapir PET and subsequent imaging with [18F]APN‐1607 in a proof‐of‐concept study and a test‐retest study, respectively, participated in the current study. Dynamic imaging with [18F]APN‐1701 was performed over 180 min to obtain kinetic data.ResultOverall, for both subjects [18F]APN‐1701 generally matched [18F]APN‐1607 findings, i.e., peak SUV values reached similar levels. The CN subject, who was amyloid negative, had slight, uniform cortical [18F]‐APN‐1701 uptake and more substantial uptake in the pallidum, pons and midbrain. SUVr images for the AD subject showed retention in similar brain regions for both tau tracers with the same rank order of regional SUV/SUVr. Notably, for this subject [18F]APN‐1701 continued to accumulate up to the end of image acquisition.Conclusion[18F]APN‐1701 demonstrates cortical uptake generally similar to [18F]APN‐1607 but its slow kinetics are unfavorable for further development.

Subcortical tau‐accumulation predicts neuronal dysfunction in the cortex based on functional connectivity in 4R‐tauopathies

AbstractBackground4‐repeat (4R) tauopathies are neurodegenerative diseases characterized by cerebral accumulation of 4R tau pathology. The most prominent 4R‐tauopathies are progressive‐supranuclear‐palsy (PSP) and corticobasal‐syndrome (CBS) characterized by tau accumulation in subcortical nuclei as well as cortical neuronal dysfunction, as shown by PET‐assessed hypoperfusion and glucose hypometabolism. Yet, there is a spatial mismatch between subcortical tau deposition patterns and cortical neuronal dysfunction and it is unclear how these two pathological brain changes are interrelated. Here, we hypothesized that subcortical tau pathology induces diaschisis‐like neuronal dysfunction in functionally connected cortical regions.MethodWe included 47 patients with clinically diagnosed PSP or CBS who underwent structural MRI and 18F‐PI‐2620 tau‐PET. PI‐2620 PET was recorded using a dynamic one‐shot, two‐stop acquisition protocol, to determine an early 0.5‐2.5min post‐tracer‐injection perfusion window for assessing cortical neuroinjury in 200 cortical ROIs of the Schaefer atlas, as well as a 20‐40min post‐tracer‐injection window to determine 4R‐tau load in 32 subcortical ROIs of the TIAN atlas. We determined tau epicenters as 10% of subcortical ROIs with highest tau‐PET, and assessed the connectivity of tau epicenters to cortical ROIs using an age‐matched 3T resting‐state fMRI template derived from 69 healthy elderly. Using linear regression, we assessed whether i) higher subcortical tau‐PET was associated with overall reduced cortical perfusion and ii) whether cortical hypoperfusion was observed preferentially in regions closely connected to subcortical tau epicenters.ResultAs hypothesized, higher subcortical tau‐PET was associated with lower cortical perfusion (R=‐0,37, p‐value: <0,011, Fig.1). Using group‐average tau‐PET and perfusion‐PET, we found that the seed‐based connectivity pattern of subcortical tau epicenters predicted cortical perfusion patterns, where cortical regions that were more closely connected to the tau epicenter showed stronger hypoperfusion (R=‐0,16, p‐value: <0,023, Fig.2A). This association was also observed on the subject level, as indicated by overall negative b‐values of the association between tau epicenter connectivity and cortical perfusion (one‐sample t‐test: t‐value: ‐3,45, p‐value: <0,001, Fig.3).ConclusionIn 4R‐tauopathies subcortical tau‐accumulation is associated with remote neuronal dysfunction in functionally connected cortical regions. This suggests that subcortical tau pathology may induce diaschisis‐like cortical dysfunction, which may contribute to clinical disease manifestation and clinical heterogeneity.

Discriminative binding of tau PET tracers PI2620, MK6240 and RO948 in Alzheimer’s disease, corticobasal degeneration and progressive supranuclear palsy brains

Recent mechanistic and structural studies have challenged the classical tauopathy classification approach and revealed the complexity and heterogeneity of tau pathology in Alzheimer’s disease (AD) and primary tauopathies such as corticobasal degeneration (CBD) and progressive supranuclear palsy (PSP), progressing beyond distinct tau isoforms. In this multi-tau tracer study, we focused on the new second-generation tau PET tracers PI2620, MK6240 and RO948 to investigate this tau complexity in AD, CBD, and PSP brains using post-mortem radioligand binding studies and autoradiography of large and small frozen brain sections. Saturation binding studies indicated multiple binding sites for 3H-PI2620 in AD, CBD and PSP brains with different binding affinities (Kd ranging from 0.2 to 0.7 nM) and binding site densities (following the order: BmaxAD > BmaxCBD > BmaxPSP). Competitive binding studies complemented these findings, demonstrating the presence of two binding sites [super-high affinity (SHA): IC50(1) = 8.1 pM; and high affinity (HA): IC50(2) = 4.9 nM] in AD brains. Regional binding distribution studies showed that 3H-PI2620 could discriminate between AD (n = 6) and control cases (n = 9), especially in frontal cortex and temporal cortex tissue (p < 0.001) as well as in the hippocampal region (p = 0.02). 3H-PI2620, 3H-MK6240 and 3H-RO948 displayed similar binding behaviour in AD brains (in both homogenate competitive studies and one large frozen hemispherical brain section autoradiography studies) in terms of binding affinities, number of sites and regional patterns. Our small section autoradiography studies in the frontal cortex of CBD (n = 3) and PSP brains (n = 2) showed high specificity for 3H-PI2620 but not for 3H-MK6240 or 3H-RO948. Our findings clearly demonstrate different binding properties among the second-generation tau PET tracers, which may assist in further understanding of tau heterogeneity in AD versus non-AD tauopathies and suggests potential for development of pure selective 4R tau PET tracers.

Quantitative proteomics of tau and Aβ in detergent fractions from Alzheimer's disease brains.

The two hallmarks of Alzheimer's disease (AD) are amyloid-β (Aβ) plaques and neurofibrillary tangles marked by phosphorylated tau. Increasing evidence suggests that aggregating Aβ drives tau accumulation, a process that involves synaptic degeneration leading to cognitive impairment. Conversely, there is a realization that non-fibrillar (oligomeric) forms of Aβ mediate toxicity in AD. Fibrillar (filamentous) aggregates of proteins across the spectrum of the primary and secondary tauopathies were the focus of recent structural studies with a filament structure-based nosologic classification, but less emphasis was given to non-filamentous co-aggregates of insoluble proteins in the fractions derived from post-mortem human brains. Here, we revisited sarkosyl-soluble and -insoluble extracts to characterize tau and Aβ species by quantitative targeted mass spectrometric proteomics, biochemical assays, and electron microscopy. AD brain sarkosyl-insoluble pellets were greatly enriched with Aβ42 at almost equimolar levels to N-terminal truncated microtubule-binding region (MTBR) isoforms of tau with multiple site-specific post-translational modifications (PTMs). MTBR R3 and R4 tau peptides were most abundant in the sarkosyl-insoluble materials with a 10-fold higher concentration than N-terminal tau peptides. This indicates that the major proportion of the enriched tau was the aggregation-prone N-terminal and proline-rich region (PRR) of truncated mixed 4R and 3R tau with more 4R than 3R isoforms. High concentration and occupancies of site-specific phosphorylation pT181 (~22%) and pT217 (~16%) (key biomarkers of AD) along with other PTMs in the PRR and MTBR indicated a regional susceptibility of PTMs in aggregated tau. Immunogold labelling revealed that tau may exist in globular non-filamentous form (N-terminal intact tau) co-localized with Aβ in the sarkosyl-insoluble pellets along with tau filaments (N-truncated MTBR tau). Our results suggest a model that Aβ and tau interact forming globular aggregates, from which filamentous tau and Aβ emerge. These characterizations contribute towards unravelling the sequence of events which lead to end-stage AD changes.

A 3D-induced pluripotent stem cell-derived human neural culture model to study certain molecular and biochemical aspects of Alzheimer’s disease

PurposeAlzheimer’s disease (AD) early pathology needs better understanding and models. Here, we describe a human induced pluripotent stem cells (iPSCs)-derived 3D neural culture model to study certain aspects of AD biochemistry and pathology.MethodiPSCs derived from controls and AD patients with Presenilin1 mutations were cultured in a 3D platform with a similar microenvironment to the brain, to differentiate into neurons and astrocytes and self-organise into 3D structures by 3 weeks of differentiation in vitro.ResultsCells express astrocytic (GFAP), neuronal (β3-Tubulin, MAP2), glutamatergic (VGLUT1), GABAergic (GAD65/67), pre-synaptic (Synapsin1) markers and a low level of neural progenitor cell (Nestin) marker after 6 and 12 weeks of differentiation in 3D. The foetal 3R Tau isoforms and adult 4R Tau isoforms were detected at 6 weeks post differentiation, showing advanced neuronal maturity. In the 3D AD cells, total and insoluble Tau levels were higher than in 3D control cells.ConclusionOur data indicates that this model may recapitulate the early biochemical and pathological disease features and can be a relevant platform for studying early cellular and biochemical changes and the identification of drug targets.

SMaRT modulation of tau isoforms rescues cognitive and motor impairments in a preclinical model of tauopathy.

Tau is a microtubule-associated protein predominantly expressed in neurons, which participates in microtubule polymerization and axonal transport. Abnormal tau metabolism leads to neurodegenerative diseases named tauopathies, such as Alzheimer’s disease and frontotemporal dementia. The alternative splicing of exon 10 (E10) in the primary transcript produces tau protein isoforms with three (3R) or four (4R) microtubule binding repeats, which are found in equal amounts in the normal adult human brain. Several tauopathies are associated with abnormal E10 alternative splicing, leading to an imbalance between 3R and 4R isoforms, which underlies disease. Correction of such imbalance represents a potential disease-modifying therapy for those tauopathies. We have previously optimized a trans-splicing RNA reprogramming strategy to modulate the 3R:4R tau content in a mouse model of tauopathy related to tau mis-splicing (htau mice), and showed that local modulation of E10 inclusion in the prefrontal cortex prevents cognitive decline, neuronal firing impairments and hyperphosphorylated tau accumulation. Furthermore, local shifting of 3R–4R tau into the striatum of htau mice prevented motor coordination deficits. However, a major bottleneck of our previous work is that local splicing regulation was performed in young mice, before the onset of pathological phenotypes. Here we tested whether regulation of tau E10 splicing could rescue tau pathology phenotypes in htau mice, after the onset of cognitive and motor impairments, comparable to early stages of human tauopathies. To determine phenotypic time course and affected brain nuclei, we assessed htau mice using behavioural tests and microPET FDG imaging over time, similarly to diagnosis methods used in patients. Based on these analyses, we performed local delivery of pre-trans splicing molecules to regulate E10 inclusion either into the medial prefrontal cortex (mPFC) or the striatum at 6-month-old once behavioral phenotypes and metabolic changes were detected. Tau isoforms modulation into the mPFC restored cognitive performance in mice that previously showed mild to severe memory impairment while motor coordination deficit was rescued after striatal injection of trans-splicing molecules. Our data suggest that tau regulation could recover pathological phenotypes early after phenotypic onset, raising promising perspectives for the use of RNA based therapies in tauopathies related to MAPT abnormal splicing.

Patients carrying the mutation p.R406W in MAPT present with non-conforming phenotypic spectrum.

The missense mutation p.R406W in microtubule-associated protein tau leads to frontotemporal lobar degeneration with an amnestic, Alzheimer's disease-like phenotype with an autosomal dominant pattern of inheritance. In 2003, we described the pedigree of a Belgian family, labelled ADG, with 28 p.R406W patients. Over 18 years follow-up, we extended the family with 10 p.R406W carriers and provided an in-depth clinical description of the patients. Additionally, genetic screening was used to identify p.R406W carriers in Belgian cohorts of frontotemporal dementia and Alzheimer's disease patients and to calculate p.R406W frequency. In the frontotemporal dementia cohort, we found four p.R406W carriers (n = 647, 0.62%) and three in the Alzheimer's disease cohort (n = 1134, 0.26%). Haplotype sharing analysis showed evidence of a shared haplotype suggesting that they are descendants of a common ancestor. Of the p.R406W patients, we describe characteristics of neuropsychological, imaging and fluid biomarkers as well as neuropathologic examination. Intriguingly, the phenotypic spectrum among the p.R406W patients ranged from typical behavioural variant frontotemporal dementia to clinical Alzheimer's disease, based on CSF biomarker analysis and amyloid PET scan. Heterogeneous overlap syndromes existed in between, with highly common neuropsychiatric symptoms like disinhibition and aggressiveness, which occurred in 100% of frontotemporal dementia and 58% of clinical Alzheimer's disease patients. This was also the case for memory problems, 89% in frontotemporal dementia and 100% in clinical Alzheimer's disease patients. Median age at death was significantly lower in patients with frontotemporal dementia (68 years) compared to clinical Alzheimer's disease patients (79 years), although the sizes of the sub-cohorts are limited and do not allow prognostic predictions. Post-mortem brain analysis of one p.R406W patient with behavioural variant frontotemporal dementia revealed frontotemporal lobar degeneration with tau pathology. Notably, neuropathological investigation showed only 3R tau isoforms in the absence of 4R tau reactivity, an unusual finding in microtubule-associated protein tau-related frontotemporal lobar degeneration. No traces of amyloid pathology were present. Prevalence of the p.R406W mutation was relatively high in both frontotemporal dementia and Alzheimer's disease Belgian patient cohorts. These findings grant new insights into genotype-phenotype correlations of p.R406W carriers. They may help in further unravelling of the pathophysiology of this tauopathy and in facilitating the identification of patients with p.R406W-related frontotemporal lobar degeneration, both in clinical diagnostic and research settings.

Evaluation of Tau Radiotracers in Chronic Traumatic Encephalopathy.

Chronic traumatic encephalopathy (CTE) is a neurologic disorder associated with head injuries, diagnosed by the perivascular accumulation of hyperphosphorylated tau protein (phospho-tau) identified at autopsy. Tau PET radiopharmaceuticals developed for imaging Alzheimer disease are under evaluation for brain injuries. The goal of this study was to conduct a head-to-head invitro evaluation of 5 tau PET radiotracers in subjects pathologically diagnosed with CTE. Methods: Autoradiography was used to assess the specific binding and distribution of 3H-flortaucipir (also known as Tauvid, AV-1451, and T807), 3H-MK-6240 (also known as florquinitau), 3H-PI-2620, 3H-APN-1607 (also known as PM-PBB3 and florzolotau), and 3H-CBD-2115 (also known as 3H-OXD-2115) in fresh-frozen human postmortem CTE brain tissue (stages I-IV). Immunohistochemistry was performed for phospho-tau with AT8, 3R tau with RD3, 4R tau with RD4 and amyloid-β with 6F/3D antibodies. Tau target density (maximum specific binding) was quantified by saturation analysis with 3H-flortaucipir in tissue sections. Results: 3H-flortaucipir demonstrated a positive signal in all CTE cases examined, with varying degrees of specific binding (68.7% ± 10.5%; n = 12) defined by homologous blockade and to a lesser extent by heterologous blockade with MK-6240 (27.3% ± 13.6%; n = 12). The 3H-flortaucipir signal was also displaced by the monoamine oxidase (MAO)-A inhibitor clorgyline (43.9% ± 4.6%; n = 3), indicating off-target binding to MAO-A. 3H-APN-1607 was moderately displaced in homologous blocking studies and was not displaced by 3H-flortaucipir; however, substantial displacement was observed when blocking with the β-amyloid-targeting compound NAV-4694. 3H-MK-6240 and 3H-PI-2620 had negligible binding in all but 2 CTE IV cases, and binding may be attributed to pathology severity or mixed Alzheimer disease/CTE pathology. 3H-CBD-2115 showed moderate binding, displaced under homologous blockade, and aligned with 4R-tau immunostaining. Conclusion: In human CTE tissues, 3H-flortaucipir and 3H-APN-1607 revealed off-target binding to MAO-A and amyloid-β, respectively, and should be considered if these radiotracers are used in PET imaging studies of patients with brain injuries. 3H-MK-6240 and 3H-PI-2620 bind to CTE tau in severe- or mixed-pathology cases, and their respective 18F PET radiotracers warrant further evaluation in patients with severe suspected CTE.

Isoform-specific patterns of tau burden and neuronal degeneration in MAPT-associated frontotemporal lobar degeneration

Frontotemporal lobar degeneration with MAPT pathogenic variants (FTLD-MAPT) has heterogeneous tau pathological inclusions postmortem, consisting of three-repeat (3R) or four-repeat (4R) tau isoforms, or a combination (3R + 4R). Here, we studied grey matter tau burden, its relation to neuronal degeneration, and regional patterns of pathology in different isoform groups of FTLD-MAPT. We included 38 FTLD-MAPT autopsy cases with 10 different MAPT pathogenic variants, grouped based on predominant tau isoform(s). In up to eleven regions (ten cortical and one striatal), we quantified grey matter tau burden using digital histopathological analysis and assigned semi-quantitative ratings for neuronal degeneration (i.e. 0–4) and separate burden of glial and neuronal tau inclusions (i.e. 0–3). We used mixed modelling to compare pathology measures (1) across the entire cohort and (2) within isoform groups. In the total cohort, tau burden and neuronal degeneration were positively associated and most severe in the anterior temporal, anterior cingulate and transentorhinal cortices. Isoform groups showed distinctive features of tau burden and neuronal degeneration. Across all regions, the 3R isoform group had lower tau burden compared to the 4R group (p = 0.008), while at the same time showing more severe neuronal degeneration than the 4R group (p = 0.002). The 3R + 4R group had an intermediate profile with relatively high tau burden along with relatively severe neuronal degeneration. Neuronal tau inclusions were most frequent in the 4R group (p < 0.001 vs. 3R), while cortical glial tau inclusions were most frequent in the 3R + 4R and 4R groups (p ≤ 0.009 vs. 3R). Regionally, neuronal degeneration was consistently most severe in the anterior temporal cortex within each isoform group. In contrast, the regions with the highest tau burden differed in isoform groups (3R: striatum; 3R + 4R: striatum, inferior parietal lobule, middle frontal cortex, anterior cingulate cortex; 4R: transentorhinal cortex, anterior temporal cortex, fusiform gyrus). We conclude that FTLD-MAPT isoform groups show distinctive features of overall neuronal degeneration and regional tau burden, but all share pronounced anterior temporal neuronal degeneration. These data suggest that distinct isoform-related mechanisms of genetic tauopathies, with slightly divergent tau distribution, may share similar regional vulnerability to neurodegeneration within the frontotemporal paralimbic networks.

A Machine Learning-Based Approach to Discrimination of Tauopathies Using [18 F]PM-PBB3 PET Images.

We recently developed a positron emission tomography (PET) probe, [18 F]PM-PBB3, to detect tau lesions in diverse tauopathies, including mixed three-repeat and four-repeat (3R + 4R) tau fibrils in Alzheimer's disease (AD) and 4R tau aggregates in progressive supranuclear palsy (PSP). For wider availability of this technology for clinical settings, bias-free quantitative evaluation of tau images without a priori disease information is needed. We aimed to establish tau PET pathology indices to characterize PSP and AD using a machine learning approach and test their validity and tracer capabilities. Data were obtained from 50 healthy control subjects, 46 patients with PSP Richardson syndrome, and 37 patients on the AD continuum. Tau PET data from 114 regions of interest were subjected to Elastic Net cross-validation linear classification analysis with a one-versus-the-rest multiclass strategy to obtain a linear function that discriminates diseases by maximizing the area under the receiver operating characteristic curve. We defined PSP- and AD-tau scores for each participant as values of the functions optimized for differentiating PSP (4R) and AD (3R + 4R), respectively, from others. The discriminatory ability of PSP- and AD-tau scores assessed as the area under the receiver operating characteristic curve was 0.98 and 1.00, respectively. PSP-tau scores correlated with the PSP rating scale in patients with PSP, and AD-tau scores correlated with Mini-Mental State Examination scores in healthy control-AD continuum patients. The globus pallidus and amygdala were highlighted as regions with high weight coefficients for determining PSP- and AD-tau scores, respectively. These findings highlight our technology's unbiased capability to identify topologies of 3R + 4R versus 4R tau deposits. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Recapitulation of endogenous 4R tau expression and formation of insoluble tau in directly reprogrammed human neurons.

Tau is a microtubule-binding protein expressed in neurons, and the equal ratios between 4-repeat (4R) and 3-repeat (3R) isoforms are maintained in normal adult brain function. Dysregulation of 3R:4R ratio causes tauopathy, and human neurons that recapitulate tau isoforms in health and disease will provide a platform for elucidating pathogenic processes involving tau pathology. We carried out extensive characterizations of tau isoforms expressed in human neurons derived by microRNA-induced neuronal reprogramming of adult fibroblasts. Transcript and protein analyses showed that miR neurons expressed all six isoforms with the 3R:4R isoform ratio equivalent to that detected in human adult brains. Also, miR neurons derived from familial tauopathy patients with a 3R:4R ratio altering mutation showed increased 4R tau and the formation of insoluble tau with seeding activity. Our results collectively demonstrate the utility of miRNA-induced neuronal reprogramming to recapitulate endogenous tau regulation comparable with the adult brain in health and disease.

Fluent molecular mixing of Tau isoforms in Alzheimer’s disease neurofibrillary tangles

Alzheimer’s disease (AD) is defined by intracellular neurofibrillary tangles formed by the microtubule-associated protein tau and extracellular plaques formed by the β-amyloid peptide. AD tau tangles contain a mixture of tau isoforms with either four (4R) or three (3R) microtubule-binding repeats. Here we use solid-state NMR to determine how 4R and 3R tau isoforms mix at the molecular level in AD tau aggregates. By seeding differentially isotopically labeled 4R and 3R tau monomers with AD brain-derived tau, we measured intermolecular contacts of the two isoforms. The NMR data indicate that 4R and 3R tau are well mixed in the AD-tau seeded fibrils, with a 60:40 incorporation ratio of 4R to 3R tau and a small homotypic preference. The AD-tau templated 4R tau, 3R tau, and mixed 4R and 3R tau fibrils exhibit no structural differences in the rigid β-sheet core or the mobile domains. Therefore, 4R and 3R tau are fluently recruited into the pathological fold of AD tau aggregates, which may explain the predominance of AD among neurodegenerative disorders.

Detection of astrocytic tau pathology facilitates recognition of chronic traumatic encephalopathy neuropathologic change

Traumatic brain injury (TBI) is associated with the development of a range of neurodegenerative pathologies, including chronic traumatic encephalopathy (CTE). Current consensus diagnostic criteria define the pathognomonic cortical lesion of CTE neuropathologic change (CTE-NC) as a patchy deposition of hyperphosphorylated tau in neurons, with or without glial tau in thorn-shaped astrocytes, typically towards the depths of sulci and clustered around small blood vessels. Nevertheless, although incorporated into consensus diagnostic criteria, the contribution of the individual cellular components to identification of CTE-NC has not been formally evaluated. To address this, from the Glasgow TBI Archive, cortical tissue blocks were selected from consecutive brain donations from contact sports athletes in which there was known to be either CTE-NC (n = 12) or Alzheimer’s disease neuropathologic change (n = 4). From these tissue blocks, adjacent tissue sections were stained for tau antibodies selected to reveal either solely neuronal pathology (3R tau; GT-38) or mixed neuronal and astroglial pathologies (4R tau; PHF-1). These stained sections were then randomised and independently assessed by a panel of expert neuropathologists, blind to patient clinical history and primary antibody applied to each section, who were asked to record whether CTE-NC was present. Results demonstrate that, in sections stained for either 4R tau or PHF-1, consensus recognition of CTE-NC was high. In contrast, recognition of CTE-NC in sections stained for 3R tau or GT-38 was poor; in the former no better than chance. Our observations demonstrate that the presence of both neuronal and astroglial tau pathologies facilitates detection of CTE-NC, with its detection less consistent when neuronal tau pathology alone is visible. The combination of both glial and neuronal pathologies, therefore, may be required for detection of CTE-NC.

Author Response: Progressive Auditory Verbal Agnosia Secondary to Alzheimer Disease

We appreciate the interest in our case.1 The patient has both amyloid (PiB positive) and tau in her brain, which would be consistent with a biological definition of Alzheimer disease. Tau PET generally binds to 3R and 4R tau of Alzheimer and does not bind to 3R or 4R tauopathies, individually. However, we cannot exclude the possibility of a second pathologic process and agree that longitudinal follow-up, repeat biomarkers, and pathology will be important for this case.

Astrocytic 4R tau expression drives astrocyte reactivity and dysfunction.

The protein tau and its isoforms are associated with several neurodegenerative diseases, many of which are characterized by greater deposition of the 4-repeat (4R) tau isoform; however, the role of 4R tau in disease pathogenesis remains unclear. We created antisense oligonucleotides (ASOs) that alter the ratio of 3R to 4R tau to investigate the role of specific tau isoforms in disease. Preferential expression of 4R tau in human tau–expressing (hTau-expressing) mice was previously shown to increase seizure severity and phosphorylated tau deposition without neuronal or synaptic loss. In this study, we observed strong colocalization of 4R tau within reactive astrocytes and increased expression of pan-reactive and neurotoxic genes following 3R to 4R tau splicing ASO treatment in hTau mice. Increasing 4R tau levels in primary astrocytes provoked a similar response, including a neurotoxic genetic profile and diminished homeostatic function, which was replicated in human induced pluripotent stem cell–derived (iPSC-derived) astrocytes harboring a mutation that exhibits greater 4R tau. Healthy neurons cultured with 4R tau–expressing human iPSC–derived astrocytes exhibited a higher firing frequency and hypersynchrony, which could be prevented by lowering tau expression. These findings support a potentially novel pathway by which astrocytic 4R tau mediates reactivity and dysfunction and suggest that astrocyte-targeted therapeutics against 4R tau may mitigate neurodegenerative disease progression.

Distinct populations of highly potent TAU seed conformers in rapidly progressing Alzheimer's disease.

Although genetic factors play a main role in determining the risk of developing Alzheimer’s disease (AD), they do not explain extensive spectrum of clinicopathological phenotypes. Deposits of aggregated TAU proteins are one of the main predictors of cognitive decline in AD. We investigated the hypothesis that variabilities in AD progression could be due to diverse structural assemblies (strains) of TAU protein. Using sensitive biophysical methods in 40 patients with AD and markedly different disease durations, we identified populations of distinct TAU particles that differed in size, structural organization, and replication rate in vitro and in cell assay. The rapidly replicating, distinctly misfolded TAU conformers found in rapidly progressive AD were composed of ~80% misfolded four-repeat (4R) TAU and ~20% of misfolded 3R TAU isoform with the same conformational signatures. These biophysical observations suggest that distinctly misfolded population of 4R TAU conformers drive the rapid decline in AD and imply that effective therapeutic strategies might need to consider not a singular species but a cloud of differently misfolded TAU conformers.

Acetylation of Tau Induces Alzheimer's Disease-Associated Tau in Transgenic Mice

Alzheimer’s disease (AD) is a neurodegenerative disorder that is characterized by neurofibrillary tangles (NFTs) and amyloid beta plaques. These NFTs are made up of aggregated tau proteins. Tau is involved in stabilizing microtubules and does not usually display aggregation. Acetylation of tau protein causes an increase in tau aggregation but its role in AD progression is still not well understood. I hypothesized that enhanced acetylated tau results in an increase in AD-like tau pathology. To test this, a murine prion promoter-tauKQ transgene was injected into the mouse fertilized oocyte. The tauKQ mutation alters lysine to glutamine to mimic acetylation of tau. Nontransgenic mice were used as controls. AT8 and GT-38 antibodies were used in immunohistochemistry (IHC) to target phosphorylated tau and AD-associated tau, respectively. GT-38 is conformation-dependent and requires 3R and 4R tau isoforms which makes it specific to AD. Through immunofluorescence, increased phosphorylated tau was observed in the hippocampus of the tauKQ mice compared to the nontransgenic mice. I focused on the dentate gyrus, CA1 region, and the mossy fibers of the CA3 region since they are involved in many memory processes. Through chromogenic IHC, the tauKQ mice exhibited more 3R+4R tau isoform pathology in the mossy fibers than the nontransgenic mice. This data suggests that an acetylation mimic is sufficient to stimulate an abundance of AD-related tau pathology in transgenic mice which is consistent with my hypothesis. The tauKQ mouse model can assist in understanding the role of tau acetylation and tau progression for AD.

Defective proteostasis in patient-derived iPSC astrocytes and neurons carrying a MAPT IVS10+16 mutation.

Impaired proteostasis is associated with normal aging and is accelerated in neurodegeneration. This impairment may lead to the toxic accumulation of protein. In a subset of frontotemporal dementia (FTD) cases, mutations in the microtubule-associated protein tau (MAPT) that alter the relative levels of tau isoforms are sufficient to cause tau inclusions in neurons and astroglia and neurodegeneration without the presence of mutated protein (e.g. MAPTIVS10+16). However, the pathogenic events triggered by the expression of the alternatively spliced tau remain poorly understood. To determine whether altered tau splicing induced from MAPT IVS10+16 mutations is sufficient to alter proteostasis in neurons and glia, we used human induced pluripotent stem cell (iPSC)-derived neurons and astrocytes from patients carrying the MAPT IVS10+16 mutation and CRISPR/Cas9, isogenic corrected controls. We found that neurons from MAPT IVS10+16 carriers exhibited significantly higher levels of tau containing 4 microtubule binding repeats (4R tau), deficits in lysosomal trafficking, and acidity relative to isogenic-control neurons. Conversely, astrocytes from MAPT IVS10+16 carriers exhibited morphologically an increase in acidic lysosomes compared to isogenic-control astrocytes. Astrocytes from MAPT IVS10+16 carriers were also larger in size, consistent with cellular hypertrophy observed in brains from FTD-tau patients. Our findings suggest that altered tau splicing induced by the MAPT IVS10+16 mutation is sufficient to cause impaired lysosomal function and altered proteostasis in a cell-type specific manner.