Alzheimer Neurofibrillary Tangles Research Articles

Tau phosphorylation affects its axonal transport and degradation

Phosphorylated forms of microtubule-associated protein tau accumulate in neurofibrillary tangles in Alzheimer's disease. To investigate the effects of specific phosphorylated tau residues on its function, wild type or phosphomutant tau was expressed in cells. Elevated tau phosphorylation decreased its microtubule binding and bundling, and increased the number of motile tau particles, without affecting axonal transport kinetics. In contrast, reducing tau phosphorylation enhanced the amount of tau bound to microtubules and inhibited axonal transport of tau. To determine whether differential tau clearance is responsible for the increase in phosphomimic tau, we inhibited autophagy in neurons which resulted in a 3-fold accumulation of phosphomimic tau compared with wild type tau, and endogenous tau was unaffected. In autophagy-deficient mouse embryonic fibroblasts, but not in neurons, proteasomal degradation of phosphomutant tau was also reduced compared with wild type tau. Therefore, autophagic and proteasomal pathways are involved in tau degradation, with autophagy appearing to be the primary route for clearing phosphorylated tau in neurons. Defective autophagy might contribute to the accumulaton of tau in neurodegenerative diseases.

Negative Regulation of 26S Proteasome Stability via Calpain-mediated Cleavage of Rpn10 Subunit upon Mitochondrial Dysfunction in Neurons

Proteasomal and mitochondrial dysfunctions are implicated in chronic neurodegenerative diseases. To investigate the impact of mitochondrial impairment on the proteasome, we treated rat cerebral cortical neurons with oligomycin, antimycin, or rotenone, which inhibit different elements of the electron transport chain. Firstly, we observed a reduction in ubiquitinated proteins and E1 activity. Secondly, we established that 26S proteasomes are disassembled with a decline in activity. Thirdly, we show, to our knowledge for the first time, that calpain activation triggers the selective processing of the 26S proteasome subunit Rpn10. Other proteasome subunits tested were not affected. Calpain also cleaved caspase 3 to an inactive fragment, thus preventing apoptosis that is an energy-dependent cell death pathway. In addition, calpain cleaved the microtubule-associated protein Tau, a major component of neurofibrillary tangles in Alzheimer disease and other tauopathies. Fourthly, we detected a rise in 20S proteasome levels and activity. Finally, we show that both acute (16 h) and long term (up to 7 days) mitochondrial impairment led to down-regulation of ubiquitinated-proteins, 26S proteasome disassembly, and a rise in 20S proteasomes. We postulate that upon mitochondrial dysfunction, ATP depletion and calpain activation contribute to the demise of protein turnover by the ubiquitin/proteasome pathway. The concomitant rise in 20S proteasomes, which seem to degrade proteins in an unregulated and energy-independent manner, in the short term may carry out the turnover of randomly unfolded oxidized proteins. However, if chronic, it could lead to neurodegeneration as regulated protein degradation by the ubiquitin/proteasome pathway is essential for neuronal survival.

Synthesis and biological evaluation of novel styryl benzimidazole derivatives as probes for imaging of neurofibrillary tangles in Alzheimer’s disease

This paper describes the synthesis and biological evaluation of styrylbenzimidazole (SBIM) derivatives as agents for imaging neurofibrillary tangles (NFT) in patients with Alzheimer’s disease (AD). SBIM derivatives were prepared with 4-iodobenzene-1,2-diamine and substituted cinnamaldehydes. In binding experiments using recombinant tau and Aβ1–42 aggregates, SBIM-3 showed higher affinity for the tau aggregates than Aβ1–42 aggregates (ratio of Kd values was 2.73). In in vitro autoradiography and fluorescent staining, [125I]SBIM-3 (or SBIM-3) bound NFT in sections of AD brain tissue. In biodistribution experiments using normal mice, all [125I]SBIM derivatives showed high initial uptake into (3.20–4.11%ID/g at 2min after the injection) and rapid clearance from (0.12–0.33%ID/g at 60min after the injection) the brain. In conclusion, appropriate structural modifications of SBIM derivatives could lead to more useful agents for the in vivo imaging of NFT in AD brains.

Synthetic Tau Fibrils Mediate Transmission of Neurofibrillary Tangles in a Transgenic Mouse Model of Alzheimer's-Like Tauopathy

Tauopathies, including Alzheimer's disease (AD) and frontotemporal lobar degeneration with tau pathologies, are neurodegenerative diseases characterized by neurofibrillary tangles (NFTs) comprising filamentous tau protein. Although emerging evidence suggests that tau pathology may be transmitted, we demonstrate here that synthetic tau fibrils are sufficient to transmit tau inclusions in a mouse model. Specifically, intracerebral inoculation of young PS19 mice overexpressing mutant human tau (P301S) with synthetic preformed fibrils (pffs) assembled from recombinant full-length tau or truncated tau containing four microtubule binding repeats resulted in rapid induction of NFT-like inclusions that propagated from injected sites to connected brain regions in a time-dependent manner. Interestingly, injection of tau pffs into either hippocampus or striatum together with overlaying cortex gave rise to distinct pattern of spreading. Moreover, unlike tau pathology that spontaneously develops in old PS19 mice, the pff-induced tau inclusions more closely resembled AD NFTs because they were Thioflavin S positive, acetylated, and more resistant to proteinase K digestion. Together, our study demonstrates that synthetic tau pffs alone are capable of inducing authentic NFT-like tau aggregates and initiating spreading of tau pathology in a tauopathy mouse model.

Keap1 Is Localized in Neuronal and Glial Cytoplasmic Inclusions in Various Neurodegenerative Diseases

Oxidative stress has been proposed as a potential mechanism for neurodegenerative diseases, such as Alzheimer disease (AD), Parkinson disease (PD), and amyotrophic lateral sclerosis (ALS). In response to oxidative stress, the levels of numerous cytoprotective products are increased via alteration of the Kelch-like ECH-associated protein 1 (Keap1) and NF-E2-related factor 2 (Nrf2) system. One of the Nrf2 targets, p62, has been known to be incorporated into a wide spectrum of cytoplasmic inclusions in neurodegenerative diseases and interact with Keap1. However, it remains unclear whether Keap1 is associated with the pathogenesis of neurodegenerative diseases. In this study, we investigated the relationship between p62 and Keap1 in the brains of patients with AD, PD, dementia with Lewy bodies (DLB), and ALS. Biochemical analyses showed that p62 and Keap1 interacted with each other in AD and DLB brains and were extracted into similar detergent-soluble and -insoluble fractions. Pathologic examination demonstrated that anti-Keap1 antibodies immunostained Lewy bodies in PD and DLB, neurofibrillary tangles in AD, and skeinlike inclusions in ALS. Further analysis showed that the levels of common Nrf2 target genes were increased in AD compared with those in controls. However, there were no statistical significances in the levels of Nrf2 target genes in DLB relative to controls. Our pathologic and biochemical results suggest a molecular basis for stress response to be involved in the formation of cytoplasmic inclusions observed in several neurodegenerative diseases.

STOX1A induces phosphorylation of tau proteins at epitopes hyperphosphorylated in Alzheimer's disease

Intraneuronal fibrillary tangles are a major hallmark of several neurodegenerative diseases including Alzheimer's disease. The major constituents of these hallmarks are hyper-phosphorylated tau. In this study we used a neuronal cellular model which over-expresses transcription factor STOX1A in combination with the longest human tau isoform to test the effect of STOX1A on tau phosphorylation. Our results show that STOX1A induces phosphorylation of the longest human tau isoform at phospho-epitopes typically found in neurofibrillary tangles in Alzheimer's disease. In conclusion, our results show a STOX1A-dependent effect on tau phosphorylation found in neurodegenerative diseases such as Alzheimer's disease.

Characterization of Thorn‐Shaped Astrocytes in White Matter of Temporal Lobe in Alzheimer's Disease Brains

Thorn-shaped astrocytes (TsA) are mainly localized in the periventricular white matter of the temporal lobe in a subgroup of aged individuals usually in the context of Alzheimer's disease (AD). Immunohistochemistry of TsA shows 4Rtau deposition, tau phosphorylation at different sites recognized with phosphospecific anti-tau antibodies Thr181, Ser202, Ser214, Thr231, Ser396, Ser422, and clones AT8 and PHF-1, and conformational changes revealed with Alz50 and MC-1 antibodies; TsA are also immunostained with antibodies to active tau kinases MAPK/ERK-P, SAPK/JNK-P, p38-P and GSK-3β. These findings are common to neurofibrillary tangles in AD. However, TsA are not stained with 3Rtau antibodies, and they are seldom stained or not at all with phosphospecific tauSer262 and with Tau-C3 antibody, which recognizes the latter tau truncation at aspartic acid 421. Previous studies have shown that tau phosphorylation at Ser262 reduces tau binding to microtubules and increases caspase-3 activity, whereas tau truncation at aspartic acid 421 is associated with tau ubiquitination, and toxic effects of tau. In this line, ubiquitin is not accumulated in TsA, and in situ end-labeling of nuclear DNA fragmentation shows absence of degeneration in TsA. These observations support the concept that tau lesions in neurons differ from those seen in TsA in AD.

Predicting the location of human perirhinal cortex, Brodmann's area 35, from MRI

The perirhinal cortex (Brodmann's area 35) is a multimodal area that is important for normal memory function. Specifically, perirhinal cortex is involved in the detection of novel objects and manifests neurofibrillary tangles in Alzheimer's disease very early in disease progression. We scanned ex vivo brain hemispheres at standard resolution (1 mm × 1 mm × 1 mm) to construct pial/white matter surfaces in FreeSurfer and scanned again at high resolution (120 μm × 120 μm × 120 μm) to determine cortical architectural boundaries. After labeling perirhinal area 35 in the high resolution images, we mapped the high resolution labels to the surface models to localize area 35 in fourteen cases. We validated the area boundaries determined using histological Nissl staining. To test the accuracy of the probabilistic mapping, we measured the Hausdorff distance between the predicted and true labels and found that the median Hausdorff distance was 4.0mm for the left hemispheres (n=7) and 3.2mm for the right hemispheres (n=7) across subjects. To show the utility of perirhinal localization, we mapped our labels to a subset of the Alzheimer's Disease Neuroimaging Initiative dataset and found decreased cortical thickness measures in mild cognitive impairment and Alzheimer's disease compared to controls in the predicted perirhinal area 35. Our ex vivo probabilistic mapping of the perirhinal cortex provides histologically validated, automated and accurate labeling of architectonic regions in the medial temporal lobe, and facilitates the analysis of atrophic changes in a large dataset for earlier detection and diagnosis.

Synthesis and biological evaluation of novel oxindole derivatives for imaging neurofibrillary tangles in Alzheimer’s disease

This letter describes the synthesis and biological evaluation of a novel series of radioiodinated oxindole (OI) derivatives for detecting neurofibrillary tangles (NFTs) in the brains of patients with Alzheimer’s disease (AD). In binding experiments in vitro, 2-oxindole (2-OI) and 3-oxindole (3-OI) derivatives showed affinity for tau aggregates. The 3-OI derivative 14 showed the highest affinity of these derivatives. In biodistribution experiments using normal mice, the OI derivatives displayed good uptake (2.4–2.5%ID/g at 2min) and clearance from the brain with time (0.6–1.4%ID/g at 30min). In fluorescence staining experiments using AD brain sections, 14 clearly stained NFTs. 3-OI may serve as a new molecular scaffold for developing novel NFT imaging agents.

Discovery of Novel Potent and Highly Selective Glycogen Synthase Kinase-3β (GSK3β) Inhibitors for Alzheimer’s Disease: Design, Synthesis, and Characterization of Pyrazines

Glycogen synthase kinase-3β, also called tau phosphorylating kinase, is a proline-directed serine/threonine kinase which was originally identified due to its role in glycogen metabolism. Active forms of GSK3β localize to pretangle pathology including dystrophic neuritis and neurofibrillary tangles in Alzheimer's disease (AD) brain. By using a high throughput screening (HTS) approach to search for new chemical series and cocrystallization of key analogues to guide the optimization and synthesis of our pyrazine series, we have developed highly potent and selective inhibitors showing cellular efficacy and blood-brain barrier penetrance. The inhibitors are suitable for in vivo efficacy testing and may serve as a new treatment strategy for Alzheimer's disease.

Voxelwise meta-analysis of gray matter anomalies in Alzheimer's disease and mild cognitive impairment using anatomic likelihood estimation

BackgroundMany voxel-based morphometry studies in Alzheimer's disease (AD) and mild cognitive impairment (MCI) yielded not entirely consistent results. We conducted meta-analyses of gray matter anomalies to identify robust neuroanatomical changes between them. MethodsA systematic review of voxel-based morphometry studies of patients with AD and MCI relative to healthy comparison subjects in PubMed, Embase databases from January 1995 to 29 April, 2011 was conducted. The anatomical distribution of the coordinates of gray matter differences was meta-analyzed using anatomical likelihood estimation (ALE). Separate maps of gray matter changes were constructed, and subtraction meta-analysis between AD and MCI was also performed. ResultsThirty-five AD studies and twenty-four MCI studies were included in the meta-analysis. Extensive gray matter deficits were present in the medial temporal lobe (including entorhinal cortex, hippocampus, parahippocampus, amygdala and uncus), thalamus, temporal, parietal, frontal and cingulate and insular cortices in AD. In MCI, gray matter reductions were identified in the medial temporal lobe (including entorhinal cortex, hippocampus, parahippocampus, amygdala and uncus), temporal, thalamus, and cingulate cortex. Subtraction meta-analysis found more severe gray matter deficit mainly in the left medial lobe (including parahippocampus, amygdala and hippocampus). ConclusionsOur meta-analysis identified similar distributions of neuroanatomical changes in AD and MCI, which are consistent with the hierarchical model of neuropathological alterations of neurofibrillary tangles in AD and MCI. Subtraction analysis provided evidence that the left MTL may be a neuroanatomical marker to evaluate disease progression from MCI to AD.

Synaptic Released Zinc Promotes Tau Hyperphosphorylation by Inhibition of Protein Phosphatase 2A (PP2A)

Hyperphosphorylated tau is the major component of neurofibrillary tangles in Alzheimer disease (AD), and the tangle distribution largely overlaps with zinc-containing glutamatergic neurons, suggesting that zinc released in synaptic terminals may play a role in tau phosphorylation. To explore this possibility, we treated cultured hippocampal slices or primary neurons with glutamate or Bic/4-AP to increase the synaptic activity with or without pretreatment of zinc chelators, and then detected the phosphorylation levels of tau. We found that glutamate or Bic/4-AP treatment caused tau hyperphosphorylation at multiple AD-related sites, including Ser-396, Ser-404, Thr-231, and Thr-205, while application of intracellular or extracellular zinc chelators, or blockade of zinc release by extracellular calcium omission almost abolished the synaptic activity-associated tau hyperphosphorylation. The zinc release and translocation of excitatory synapses in the hippocampus were detected, and zinc-induced tau hyperphosphorylation was also observed in cultured brain slices incubated with exogenously supplemented zinc. Tau hyperphosphorylation induced by synaptic activity was strongly associated with inactivation of protein phosphatase 2A (PP2A), and this inactivation can be reversed by pretreatment of zinc chelator. Together, these results suggest that synaptically released zinc promotes tau hyperphosphorylation through PP2A inhibition.

Optineurin immunoreactivity in neuronal nuclear inclusions of polyglutamine diseases (Huntington’s, DRPLA, SCA2, SCA3) and intranuclear inclusion body disease

Optineurin (OPTN) is a ubiquitous cytoplasmic protein with various functions including membrane trafficking and signal transduction [9]. OPTN mutations are associated with adult-onset glaucoma and familial and sporadic amyotrophic lateral sclerosis (ALS) [4]. OPTN immunoreactivity has been reported in a variety of ubiquitinated inclusions including ALS-associated neuronal cytoplasmic inclusions, neurofibrillary tangles in Alzheimer’s disease, Pick bodies in Pick disease, Lewy bodies in Parkinson’s disease and glial cytoplasmic inclusions in multiple system atrophy [3, 6]. With respect to polyglutamine diseases, neuronal nuclear inclusions (NNIs) in Huntington’s disease (HD) and spinocerebellar ataxia type 3 (SCA3) are reported to be immunonegative for OPTN [2]. However, Schwab et al. [7] have contended that NNIs in HD are immunopositive for OPTN. We report that OPTN immunoreactivity is present in NNIs in various polyglutamine diseases and intranuclear inclusion body disease (INIBD). A total of 30 post-mortem cases were utilized in the present study; these included cases of HD (n = 3), dentatorubral-pallidoluysian atrophy (DRPLA; n = 5), SCA2 (n = 1), SCA3 (n = 5), SCA6 (n = 2), SCA17 (n = 1), spinal and bulbar muscular atrophy (SBMA; n = 3), INIBD (n = 5) and normal control subjects (n = 5). The diagnoses of polyglutamine diseases were confirmed genetically and histopathologically. Immunohistochemical analysis was carried out using formalin-fixed, paraffinembedded sections from the basal ganglia in HD, SCA2 and SCA17, the pons in DRPLA and SCA3, the cerebellum in SCA6, the spinal cord in SBMA, the frontal lobe in INIBD, and the cortical and subcortical regions in control subjects. Anti-ubiquitin (1B3; MBL, Nagoya, Japan), antipolyglutamine (1C2; Chemicon, Temecula, CA, USA) and anti-human OPTN C terminus (#100000; Cayman CHEMICAL, Ann Arbor, MI, USA) [2, 7, 8] were used as primary antibodies. In controls, anti-OPTN antibody weakly immunolabeled the neuronal cytoplasm in a diffuse granular pattern, and neuronal nuclei were negative for OPTN (Fig. 1a). OPTN-immunoreactive NNIs were found in all the cases of HD, DRPLA, SCA2 and SCA3 (Fig. 1b–e). Intranuclear inclusions in neurons, but not in glial cells, in INIBD were also immunopositive for OPTN (Fig. 1f). Double immunofluorescence analyses revealed that OPTN immunoreactivity was found in 69% of NNIs in HD, 46% in DRPLA, 17% in SCA2, 55% in SCA3 and 50% in INIBD (Fig. 2). Although ubiquitinated NNIs were seen in SCA17 and SBMA, these inclusions were F. Mori (&) K. Tanji K. Wakabayashi Department of Neuropathology, Institute of Brain Science, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki 036-8562, Japan e-mail: neuropal@cc.hirosaki-u.ac.jp

Atomic Identification of Fluorescent Q-Dots on Tau-Positive Fibrils in 3D-Reconstructed Pick Bodies

Pick body disease, characterized by the presence of Pick bodies, is distinguished from neurofibrillary tangles in Alzheimer disease on the basis of their smooth, spherical shape. Quantum dots (QDs) are nanometer-scale, water-soluble fluorophores that are detectable both as a fluorescent signal by light microscopy and as electron-dense particles under electron microscopy. In this study, tau-positive Pick bodies were immunofluorescently labeled with QD nanocrystals composed of cadmium selenide for three-dimensional (3D) reconstruction and subsequently subjected to electron microscopic observation to identify QD immunolabeling on the same Pick body for comparison in detail. The identity of the QD nanocrystals, which label the tau-positive fibrils, was confirmed by the presence of both cadmium and selenium on these nanocrystals, demonstrated as parallel peaks corresponding to these atoms on energy-dispersive X-ray spot analysis under super-resolution scanning transmission electron microscopy. This confirmation of the specificity of the QD labeling through both its fluorescence and energy-dispersive X-ray spectra reinforces the reliability of the labeling. In addition, this exact comparison of the same structure by electron microscopy and 3D light microscopy demonstrates how its ultrastructural details are related to its surrounding structures on a 3D basis, providing further insights into how molecules woven into specific pathological ultrastructures are at work in situ.

In Vivo Spreading of Tau Pathology

In Vivo Spreading of Tau Pathology

Anti-inflammatory effects of physical activity in relationship to improved cognitive status in humans and mouse models of Alzheimer's disease.

Physical activity has been correlated with a reduced incidence of cognitive decline and Alzheimer's disease in human populations. Although data from intervention-based randomized trials is scarce, there is some indication that exercise may confer protection against age-related deficits in cognitive function. Data from animal models suggests that exercise, in the form of voluntary wheel running, is associated with reduced amyloid deposition and enhanced clearance of amyloid beta, the major constituent of plaques in Alzheimer's disease. Treadmill exercise has also been shown to ameliorate the accumulation of phosphorylated tau, an essential component of neurofibrillary tangles in Alzheimer's models. A common therapeutic theme arising from studies of exercise-induced neuroprotection in human populations and in animal models involves reduced inflammation in the central nervous system. In this respect, physical activity may promote neuronal resilience by reducing inflammation.

18F-Labeled Phenyldiazenyl Benzothiazole for in Vivo Imaging of Neurofibrillary Tangles in Alzheimer's Disease Brains

We synthesized and evaluated (E)-4-((6-(2-(2-(2-fluoroethoxy)ethoxy)ethoxy)benzo[d]thiazol-2-yl)diazenyl)-N,N-dimethylaniline (FPPDB) as a probe for the imaging of neurofibrillary tangles (NFTs) in patients with Alzheimer's disease (AD). In assays using thioflavin S (ThS) as a competitive ligand, FPPDB competed with ThS well and showed high affinity for both tau and Aβ1-42 aggregates (K i = 13.0 and 20.0 nM, respectively). The results of saturation binding assays also verified that FPPDB bound to both tau and Aβ1-42 aggregates with high affinity (K d = 44.8 nM and B max = 45.8 pmol/nmol protein for tau aggregates and K d = 45.4 nM and B max = 38.9 pmol/nmol protein for Aβ1-42 aggregates). Furthermore, [(18)F]FPPDB substantially labeled NFTs and senile plaques in AD brain sections but not control brain sections. In biodistribution experiments using normal mice, [(18)F]FPPDB displayed higher uptake (4.28% ID/g at 2 min postinjection) into and washout (2.53% ID/g at 60 min postinjection) from the brain with time. On the basis of the chemical structure of FPPDB, further increases in selective binding to tau aggregates may lead to the development of more useful probes for the imaging of NFTs in AD brains.

Ubiquitin is Associated with Early Truncation of Tau Protein at Aspartic Acid421 during the Maturation of Neurofibrillary Tangles in Alzheimer's Disease

Pathological processing of tau protein during the formation and maturation of neurofibrillary tangles (NFTs) includes abnormal phosphorylation, conformational changes and truncation of the C-terminus at aspartic-acid(421) (apoptotic product) and glutamic-acid(391) residues. Abnormal phosphorylation and misfolding may serve as recognition signals for ubiquitin-targeting and proteosomal processing. For this reason, we sought to determine whether ubiquitin-targeting of tau is associated with particular tau modifications that herald specific stages of NFTs maturation in the hippocampus of Alzheimer's disease cases. Using multiple tau antibodies, we found that 30% of the total load of NFTs is ubiquitin-associated. As reported previously ubiquitin immunoreactivity was associated with markers of phosphorylated tau in certain NFTs; however, a strong association was also found between ubiquitin and the earliest known truncation event at aspartic-acid(421) . These findings indicate that tau protein in the NFTs may be dually subjected to both apoptotic and proteosomal processing. By contrast ubiquitin immunoreactivity was poorly associated with truncation of tau at glutamic-acid(391) , suggesting that this proteolytic event may be independent of proteosomal activity. It would appear, therefore, that ubiquitin targeting of tau protein occurs at NFTs in the early and intermediate stages of the maturation.

The Role of Tau in Alzheimer's Disease and Related Disorders

Tau, the microtubule-associated protein, forms insoluble filaments that accumulate as neurofibrillary tangles in Alzheimer's disease (AD) and related tauopathies. Under physiological conditions, tau regulates the assembly and maintenance of the structural stability of microtubules. In the diseased brain, however, tau becomes abnormally hyperphosphorylated, which ultimately causes the microtubules to disassemble, and the free tau molecules aggregate into paired helical filaments. A large body of evidence suggests that tau hyperphosphorylation results from perturbation of cellular signaling, mainly through imbalance in the activities of different protein kinases and phosphatases. In AD, it appears that ß-amyloid peptide (Aß) plays a pivotal role in triggering this imbalance. In this review, we summarize our current understanding of the role of tau in AD and other tauopathies, and highlight key issues that need to be addressed to improve the success of developing novel therapies.

Structural Impact of Proline-Directed Pseudophosphorylation at AT8, AT100, and PHF1 Epitopes on 441-Residue Tau

The intrinsically disordered protein tau becomes excessively phosphorylated and aggregates into neurofibrillary tangles in Alzheimer's disease. To obtain insight into the structural consequences of phosphorylation, we characterized a mutant protein of tau in which epitopes recognized by Alzheimer diagnostic antibodies were mimicked by mutation to glutamic acid [AT8 (S199E, S202E, T205E), AT100 (T212E and S214E), and PHF1 (S396E and S404E)]. A large number of distance restraints obtained from NMR paramagnetic relaxation enhancement in combination with ensemble conformer calculations demonstrate that pseudophosphorylation causes an opening of the transient folding of tau. Together with previous studies on the Parkinson-related protein α-synuclein, our data indicate that networks of transient long-range interactions are common properties of intrinsically disordered proteins and that their modulation is important for aggregation.