Tau Variants Research Articles

Induced Tauopathy in a Novel 3D-Culture Model Mediates Neurodegenerative Processes: A Real-Time Study on Biochips

Tauopathies including Alzheimer’s disease represent one of the major health problems of aging population worldwide. Therefore, a better understanding of tau-dependent pathologies and consequently, tau-related intervention strategies is highly demanded. In recent years, several tau-focused therapies have been proposed with the aim to stop disease progression. However, to develop efficient active pharmaceutical ingredients for the broad treatment of Alzheimer’s disease patients, further improvements are necessary for understanding the detailed neurodegenerative processes as well as the mechanism and side effects of potential active pharmaceutical ingredients (API) in the neuronal system. In this context, there is a lack of suitable complex in vitro cell culture models recapitulating major aspects of taupathological degenerative processes in sufficient time and reproducible manner.Herewith, we describe a novel 3D SH-SY5Y cell-based, tauopathy model that shows advanced characteristics of matured neurons in comparison to monolayer cultures without the need of artificial differentiation promoting agents. Moreover, the recombinant expression of a novel highly pathologic fourfold mutated human tau variant lead to a fast and emphasized degeneration of neuritic processes. The neurodegenerative effects could be analyzed in real time and with high sensitivity using our unique microcavity array-based impedance spectroscopy measurement system. We were able to quantify a time- and concentration-dependent relative impedance decrease when Alzheimer’s disease-like tau pathology was induced in the neuronal 3D cell culture model. In combination with the collected optical information, the degenerative processes within each 3D-culture could be monitored and analyzed. More strikingly, tau-specific regenerative effects caused by tau-focused active pharmaceutical ingredients could be quantitatively monitored by impedance spectroscopy.Bringing together our novel complex 3D cell culture taupathology model and our microcavity array-based impedimetric measurement system, we provide a powerful tool for the label-free investigation of tau-related pathology processes as well as the high content analysis of potential active pharmaceutical ingredient candidates.

Evidence for a role of the rare p.A152T variant in MAPT in increasing the risk for FTD-spectrum and Alzheimer's diseases

Rare mutations in the gene encoding for tau (MAPT, microtubule-associated protein tau) cause frontotemporal dementia-spectrum (FTD-s) disorders, including FTD, progressive supranuclear palsy (PSP) and corticobasal syndrome, and a common extended haplotype spanning across the MAPT locus is associated with increased risk of PSP and Parkinson's disease. We identified a rare tau variant (p.A152T) in a patient with a clinical diagnosis of PSP and assessed its frequency in multiple independent series of patients with neurodegenerative conditions and controls, in a total of 15 369 subjects. Tau p.A152T significantly increases the risk for both FTD-s (n = 2139, OR = 3.0, CI: 1.6-5.6, P = 0.0005) and Alzheimer's disease (AD) (n = 3345, OR = 2.3, CI: 1.3-4.2, P = 0.004) compared with 9047 controls. Functionally, p.A152T (i) decreases the binding of tau to microtubules and therefore promotes microtubule assembly less efficiently; and (ii) reduces the tendency to form abnormal fibers. However, there is a pronounced increase in the formation of tau oligomers. Importantly, these findings suggest that other regions of the tau protein may be crucial in regulating normal function, as the p.A152 residue is distal to the domains considered responsible for microtubule interactions or aggregation. These data provide both the first genetic evidence and functional studies supporting the role of MAPT p.A152T as a rare risk factor for both FTD-s and AD and the concept that rare variants can increase the risk for relatively common, complex neurodegenerative diseases, but since no clear significance threshold for rare genetic variation has been established, some caution is warranted until the findings are further replicated.

Cognitive defects are reversible in inducible mice expressing pro-aggregant full-length human Tau

Neurofibrillary lesions of abnormal Tau are hallmarks of Alzheimer disease and frontotemporal dementias. Our regulatable (Tet-OFF) mouse models of tauopathy express variants of human full-length Tau in the forebrain (CaMKIIα promoter) either with mutation ΔK280 (pro-aggregant) or ΔK280/I277P/I308P (anti-aggregant). Co-expression of luciferase enables in vivo quantification of gene expression by bioluminescence imaging. Pro-aggregant mice develop synapse loss and Tau-pathology including missorting, phosphorylation and early pretangle formation, whereas anti-aggregant mice do not. We correlated hippocampal Tau pathology with learning/memory performance and synaptic plasticity. Pro-aggregant mice at 16 months of gene expression exhibited severe cognitive deficits in Morris water maze and in passive-avoidance paradigms, whereas anti-aggregant mice were comparable to controls. Cognitive impairment of pro-aggregant mice was accompanied by loss of hippocampal LTP in CA1 and CA3 areas and by a reduction of synaptic proteins and dendritic spines, although no neuronal loss was observed. Remarkably, memory and LTP recovered when pro-aggregant Tau was switched-OFF for ~4 months, Tau phosphorylation and missorting were reversed, and synapses recovered. Moreover, soluble and insoluble pro-aggregant hTau40 disappeared, while insoluble mouse Tau was still present. This study links early Tau pathology without neurofibrillary tangles and neuronal death to cognitive decline and synaptic dysfunction. It demonstrates that Tau-induced impairments are reversible after switching-OFF pro-aggregant Tau. Therefore, our mouse model may mimic an early phase of AD when the hippocampus does not yet suffer from irreversible cell death but cognitive deficits are already striking. It offers potential to evaluate drugs with regard to learning and memory performance.

Human Platelets Tau: A Potential Peripheral Marker for Alzheimer's Disease

Platelets are a major peripheral reservoir of the amyloid-β protein precursor, so they have been considered as a potential biological marker of Alzheimer's disease (AD). Here, it is demonstrated that tau protein is also present in platelets and that the levels of oligomeric species of this protein could serve as a novel and reliable biological marker for AD. Blood samples were obtained from 15 AD patients and 10 paired-age controls and platelets were separated via differential centrifugation. The purity of platelets was determined by flow cytometry and microscopy and the presence of tau was determined by immunofluorescence and immunoblots with tau specific antibodies. Immunofuorescence and immunoblot patterns of platelets were positive for tau. Immunoblots also showed the presence of high molecular weight (HMW) variants of tau that appeared to correspond to oligomeric forms of the protein. The ratio of HMW tau respect to tau monomeric species was significantly higher in AD patients than controls. The present is the first description of the presence of tau in platelets. The analysis of different tau fractions in platelets could serve as a new biological marker for AD.

The Hsp90 Kinase Co-chaperone Cdc37 Regulates Tau Stability and Phosphorylation Dynamics

The microtubule-associated protein tau, which becomes hyperphosphorylated and pathologically aggregates in a number of these diseases, is extremely sensitive to manipulations of chaperone signaling. For example, Hsp90 inhibitors can reduce the levels of tau in transgenic mouse models of tauopathy. Because of this, we hypothesized that a number of Hsp90 accessory proteins, termed co-chaperones, could also affect tau stability. Perhaps by identifying these co-chaperones, new therapeutics could be designed to specifically target these proteins and facilitate tau clearance. Here, we report that the co-chaperone Cdc37 can regulate aspects of tau pathogenesis. We found that suppression of Cdc37 destabilized tau, leading to its clearance, whereas Cdc37 overexpression preserved tau. Cdc37 was found to co-localize with tau in neuronal cells and to physically interact with tau from human brain. Moreover, Cdc37 levels significantly increased with age. Cdc37 knockdown altered the phosphorylation profile of tau, an effect that was due in part to reduced tau kinase stability, specifically Cdk5 and Akt. Conversely, GSK3β and Mark2 were unaffected by Cdc37 modulation. Cdc37 overexpression prevented whereas Cdc37 suppression potentiated tau clearance following Hsp90 inhibition. Thus, Cdc37 can regulate tau in two ways: by directly stabilizing it via Hsp90 and by regulating the stability of distinct tau kinases. We propose that changes in the neuronal levels or activity of Cdc37 could dramatically alter the kinome, leading to profound changes in the tau phosphorylation signature, altering its proteotoxicity and stability.

A novel organotypic tauopathy model on a new microcavity chip for bioelectronic label-free and real time monitoring

Herewith we developed a novel 3D in vitro Alzheimer's disease (AD) model, based on the human neuroblastoma cell line SH-SY5Y, which is well differentiated without the application of any agents. Furthermore AD-like pathological neurodegeneration can be induced by okadaic acid (OA) mediated hyperphosphorylation of the microtubule associated protein tau. Moreover, we established stable “rapid tauopathy cell lines” expressing additional EGFP-fused (enhanced green fluorescent protein) wildtype or a pathology-promoting mutant tau variant (P301L) by lentiviral transduction. For the sensitive and feasible quantitative detection of pathological effects on neuronal 3D-cultures by electrochemical impedance spectroscopy (EIS) we optimized and redesigned a microcavity array (MCA). The cellular contribution to impedance could be increased by the factor of 2.5 and the variance decreased by 40%. Using our optimized MCA and impedance measurement setup we were able to detect quantitatively an OA concentration- and time-dependent decrease of the impedance in 3D SH-SY5Y cultures. Moreover, we were able to detect and quantify distinct, AD-related effects triggered by tau-mutant (P301L) expression and hyperphosphorylation in our organotypic 3D-cultures with the help of impedance spectroscopy.

Transgenic mouse and cell culture models demonstrate a lack of mechanistic connection between endoplasmic reticulum stress and tau dysfunction.

In vivo aggregation of tau protein is a hallmark of many neurodegenerative disorders, including Alzheimer's disease (AD). Recent evidence has also demonstrated activation of the unfolded protein response (UPR), a cellular response to endoplasmic reticulum (ER) stress, in AD, although the role of the UPR in disease pathogenesis is not known. Here, three model systems were used to determine whether a direct mechanistic link could be demonstrated between tau aggregation and the UPR. The first model system used was SH-SY5Y cells, a neuronal cultured cell line that endogenously expresses tau. In this system, the UPR was activated using chemical stressors, tunicamycin and thapsigargin, but no changes in tau expression levels, solubility, or phosphorylation were observed. In the second model system, wild-type 4R tau and P301L tau, a variant with increased aggregation propensity, were heterologously overexpressed in HEK 293 cells. This overexpression did not activate the UPR. The last model system examined here was the PS19 transgenic mouse model. Although PS19 mice, which express the P301S variant of tau, display severe neurodegeneration and formation of tau aggregates, brain tissue samples did not show any activation of the UPR. Taken together, the results from these three model systems suggest that a direct mechanistic link does not exist between tau aggregation and the UPR.

Understanding the molecular basis of Alzheimer's disease using a Caenorhabditis elegans model system.

Alzheimer's disease (AD) is the major cause of dementia in the United States. At the cellular level, the brains of AD patients are characterized by extracellular dense plaques and intracellular neurofibrillary tangles whose major components are the beta-amyloid peptide and tau, respectively. The beta-amyloid peptide is a cleavage product of the amyloid precursor protein (APP); mutations in APP have been correlated with a small number of cases of familial Alzheimer's disease. APP is the canonical member of the APP family, whose functions remain unclear. The nematode Caenorhabditis elegans, one of the premier genetic workhorses, is being used in a variety of ways to address the functions of APP and determine how the beta-amyloid peptide and tau can induce toxicity. First, the function of the C. elegans APP-related gene, apl-1, is being examined. Although different organisms may use APP and related proteins, such as APL-1, in different functional contexts, the pathways in which they function and the molecules with which they interact are usually conserved. Second, components of the gamma-secretase complex and their respective functions are being revealed through genetic analyses in C. elegans. Third, to address questions of toxicity, onset of degeneration, and protective mechanisms, different human beta-amyloid peptide and tau variants are being introduced into C. elegans and the resultant transgenic lines examined. Here, we summarize how a simple system such as C. elegans can be used as a model to understand APP function and suppression of beta-amyloid peptide and tau toxicity in higher organisms.

Drosophila notal bristle as a novel assessment tool for pathogenic study of Tau toxicity and screening of therapeutic compounds

To elucidate the Tau gain-of-toxicity functional mechanism and to search for potential treatments, we overexpressed human Tau variants (hTau) in the dorsal mesothorax (notum) of Drosophila. Overexpression of Tau variants caused loss of notal bristles, and the phenotype was used for evaluating toxicity of ectopic Tau. The bristle loss phenotype was found to be highly associated with the toxicity of hyperphosphoryled Tau in flies. We have shown that the bristle loss phenotype can be rescued either by reducing Glycogen synthase kinase 3β (GSK3β)/Shaggy (Sgg) activity or overexpressing Bβ2 regulatory subunits of PP2A. Elevated expression of the Drosophila Bβ2 homolog, Twins (Tws), also alleviated neuritic dystrophy of the dorsal arborization (da) neuron caused by Tau aggregation. Additionally, lowering endogenous Tau dosage was beneficial as it ameliorated the bristle loss phenotype. Finally, the bristle loss phenotype was used to evaluate the efficacy of potential therapeutic compounds. The GSK3β inhibitor, alsterpaullone, was found to suppress toxicity of Tau in a concentration-dependent manner. The notum of Drosophila, thus, provides a new tool and insights into Tau-induced toxicity. It could also potentially assist in screening new drugs for possible therapeutic intervention.

Structural and functional changes in tau mutant mice neurons are not linked to the presence of NFTs

In the rTg4510 mouse model, expression of the mutant human tau variant P301L leads to development of neurofibrillary tangles (NFTs), neuronal death, and memory impairment, reminiscent of the pathology observed in human tauopathies. In the present study, we examined the effects of mutant tau expression on the electrophysiology and morphology of individual neurons using whole-cell patch-clamp recordings and biocytin filling of pyramidal cells in cortical slices prepared from rTg4510 (TG) and wild-type (WT) littermate mice. Among the TG cells, 42% contained a clear Thioflavin-S positive inclusion in the soma and were categorized as NFT positive (NFT+), while 58% had no discernable inclusion and were categorized as NFT negative (NFT−). The resting membrane potential ( V r) was significantly depolarized (+ 8 mV) in TG cells, and as a consequence, evoked repetitive action potential (AP) firing rates were also significantly increased. Further, single APs were significantly shorter in duration in TG cells and the depolarizing voltage deflection or “sag” evoked by hyperpolarization was significantly greater in amplitude. In addition to these functional electrophysiological changes, TG cells exhibited significant morphological alterations, including loss or significant atrophy of the apical tuft, reduced dendritic complexity and length, and reduced spine density. Importantly, NFT− and NFT+ TG cells were indistinguishable with regard to both morphological and electrophysiological properties. Our observations show that expression of mutated tau results in significant structural and functional changes in neurons, but that these changes occur independent of mature NFT formation.

Tau and Alzheimer's disease: From structure to transgenic mice and aggregation inhibitors

Tau pathology is a hallmark of AD, PD, and other tauopathies (FTDP17). Tau protein is changed in several ways (e.g. phosphorylation, missorting, aggregation), but the causes of neuronal degeneration are still uncertain. Several pathways of Tau-induced toxicity have been proposed: (1) Detachment of Tau from microtubules (by phosphorylation) with subsequent breakdown of microtubules, the tracks of axonal transport. (2) Aggregation of Tau into paired helical filaments. (3) Clustering of Tau on microtubules and inhibition of axonal transport, (4) Cleavage of Tau into toxic fragments. (5) Toxic functions of modified Tau. (6) Over- or understabilization of microtubules due to changes in alternative splicing or phosphorylation. We generated several inducible cell models and transgenic mouse models to distinguish between different effects of Tau and pathways of toxicity and to observe the response to switching Tau expression on and off again. Some cell models display traffic deficits upon expression of Tau, due to reduced activity of motor proteins moving along microtubules. Other cell models show that toxicity of Tau is induced by enhancing the beta-propensity of Tau (e.g. certain FTDP17 mutations), which in turn leads to proteolysis and aggregation, whereas “anti-aggregation” Tau variants are not toxic. The aggregation of Tau is reversible by inhibitor compounds or by switching off Tau expression. Analogous observations are made with inducible transgenic mice, where Tau variants with high beta-propensity lead to Tau aggregation, toxicity, and decay of neurons. The exogenous Tau species can even “poison” endogenous mouse Tau and lead to co-aggregation of both species. Here, too, the aggregation, toxicity, and even behavioral deficits are reversible after switching off the expression of exogenous Tau. The results show that the formation of Tau aggregates is remarkably dynamic, with a clear correlation to Tau-dependent toxicity, and that aggregation-induced effects are in principle reversible. Supported by MPG, DFG, ADDF, and EU/Memosad.

Tau Phosphorylation by cdk5 and Fyn in Response to Amyloid Peptide Aβ25–35: Involvement of Lipid Rafts

Alzheimer's disease (AD) is characterized by the accumulation of protein filaments, namely extracellular amyloid-beta (Abeta) fibrils and intracellular neurofibrillary tangles, which are composed of aggregated hyperphosphorylated tau. Tau hyperphosphorylation is the product of deregulated Ser/Thr kinases such as cdk5 and GSK3beta. In addition, tau hyperphosphorylation also occurs at Tyr residues. To find a link between Abeta and tau phosphorylation, we investigated the effects of short-term Abeta treatments on SHSY-5Y cells. We analyzed phosphorylated tau variants in lipid rafts and the possible role of Tyr18 and Ser396/404 tau phosphorylation in Abeta-induced signaling cascades. After 2 min of Abeta treatment, phospho-Tyr18-tau and its association with rafts increased. Phospho-Ser 396/404-tau became detectable in rafts after 10 min treatment, which temporally correlated with the detection of cdk5 and p35 activator in lipid rafts. To determine the role of cdk5 in tau phosphorylation at Ser396/404 in lipid rafts, we pre-incubated cells with cdk5 inhibitor roscovitine, and observed that the Abeta-induced tau phosphorylation at Ser 396/404 in rafts was abolished as well as cdk5/p35 association with rafts. These data suggest a role for cdk5 in the Abeta-promoted early events involving tau hyperphosphorylation, and their possible implications for AD pathogenesis.

S2-03-04: Role of aminopeptidase NPPS in tauopathy

Abstract not available.

S2-03-06: The role of tau in axonal transport and neurodegeneration

Abnormal changes in the properties of Tau protein are closely correlated with the progression of Alzheimer disease. They include aggregation into neurofibrillary tangles, hyperphosphorylation, missorting from the axons into the somatodendritic compartments of neurons. It is currently not well understood what causes the abnormal changes, and how they are related to the toxicity of Abeta, the decay of synapses, and the death of neurons. We have used different modes of live cell microscopy of primary neurons, adenoviral transfection of neurons with different variants of tau, and inducible transgenic mouse models of tauopathy. The study mainly focussed on three facets of Tau: (1) By obstructing the surface of microtubules, Tau is capable, in principle, of slowing down axonal transport by inhibiting the attachment of motor proteins. This is particularly visible in the form of reduced mitochondrial movement, which in turn causes an energy crisis in the neuron by reducing the ATP level. As a result, synapses disintegrate, and synaptic markers disappear. (2) Although Tau is mostly a “microtubule-associated protein” (MAP), its residence time on microtubules is only a few seconds, and thus Tau is able to move through cytosolic space by diffusion with surprising speed. This may be one of the factors that enables Tau to enter the somatodendritic compartment, once the normal sorting mechanisms break down during Alzheimer disease. (3) There is a debate on whether the aggregation of Tau as such is toxic to neurons, or whether tau-induced toxicity depends on other factors. Using inducible cell and transgenic mouse models we find that the toxicity of Tau to neurons is tightly correlated with its ability to form beta-structure and to aggregate. Thus mutants of Tau that have a high beta-propensity show rapid aggregation and high toxicity, whereas mutants that are unable to aggregate are not toxic. The findings argue that the toxicity of Tau to neurons requires a particular pathological conformation which promotes beta-structure and tau-tau interactions. Using small molecule inhibitors it may be possible to prevent the pathological conformation and thus to keep Tau functional in the neuron without aggregation.

P3-368: Misfolded truncated tau protein in the extraneuronal space: Inducer of neuronal death and inflammatory response

A pronounced increase of tau protein levels in the cerebrospinal fluid was found in most patients with Alzheimer's disease. This may reflect the increase of tau protein release from intracellular to the extracellular space. Previously it has been shown that different tau variants may induce neuronal death in the neuroblastoma cell lines. However it is not clear which tau modification is responsible for neuronal death and whether tau may also induce inflammatory response. Different recombinant truncated tau species and human tau isoforms were isolated and purified to homogeneity. The purified proteins were fractionated by gel electrophoresis and stained with Coomassie blue and silver stain or immunolabeled with anti-tau antibody DC25. The neurotoxicity was analyzed in the primary neuronal cultures using ToxiLight Non-Destructive Cytotoxicity Assay and CellTiter-Glo Luminescent Cell Viability Assay. In vitro primary rat mixed glial cultures were used for detection of tau mediated inflammatory response. NO release was evaluated measuring nitrite accumulation in the conditioned cell culture medium. Morphological changes of activated microglia were detected by immunohistochemical staining of immunomarkers CD11b/CD18 and CD68. Human misfolded truncated tau species but not full length tau isoforms displayed in vitro neurotoxic properties when incubated with primary neurons for 24h. Moreover, when rat primary mixed cultures were exposed to the misfolded tau proteins, dose dependent release of nitric oxide and adenylate kinase occurred. In parallel, microglia changed its morphology from ramified to the amoeboid, thus suggesting its activated state. Our results demonstrate that different misfolded tau species induce neuronal death and simultaneously promote inflammatory response mediated by microglia. Moreover, activated microglia underwent morphological transformation, presented full inflammatory repertoire and induced signaling pathways leading to NO release. The presented data suggest that not only intraneuronal misfolded tau trigger neurodegenerative cascade, but also extracellularly located misfolded tau is efficient promoter of neurodegeneration, neuroinflammation and neuronal death.

The β-Propensity of Tau Determines Aggregation and Synaptic Loss in Inducible Mouse Models of Tauopathy

Neurofibrillary lesions are characteristic for a group of human diseases, named tauopathies, which are characterized by prominent intracellular accumulations of abnormal filaments formed by the microtubule-associated protein Tau. The tauopathies are accompanied by abnormal changes in Tau protein, including pathological conformation, somatodendritic mislocalization, hyperphosphorylation, and aggregation, whose interdependence is not well understood. To address these issues we have created transgenic mouse lines in which different variants of full-length Tau are expressed in a regulatable fashion, allowing one to switch the expression on and off at defined time points. The Tau variants differ by small mutations in the hexapeptide motifs that control the ability of Tau to adopt a beta-structure conformation and hence to aggregate. The "pro-aggregation" mutant DeltaK280, derived from one of the mutations observed in frontotemporal dementias, aggregates avidly in vitro, whereas the "anti-aggregation" mutant DeltaK280/PP cannot aggregate because of two beta-breaking prolines. In the transgenic mice, the pro-aggregation Tau induces a pathological conformation and pre-tangle aggregation, even at low expression levels, the anti-aggregation mutant does not. This illustrates that abnormal aggregation is primarily controlled by the molecular structure of Tau in vitro and in the organism. Both variants of Tau become mislocalized and hyperphosphorylated independently of aggregation, suggesting that localization and phosphorylation are mainly a consequence of increased concentration. These pathological changes are reversible when the expression of Tau is switched off. The pro-aggregation Tau causes a strong reduction in spine synapses.

Neuronal Microtubule-associated Proteins: Insights into Their Structures and Functions

Two families of microtubule-associated proteins (MAPs), the MAP1 family and the MAP2/MAP4/tau superfamily, are found in neurons. This review describes the cellular and subcellular localizations, structural features, and in vitro properties of these MAP families. The MAP1 family consists of three members, MAP1A, MAP1B, and MAP1S. They not only show partial amino acid sequence homology, but also share a common structural property: each MAP1 polypeptide translated from the corresponding mRNA is proteolytically cleaved into two chains, which assemble into the mature MAP1 complex. MAP1A and MAP1B expression is complementary during neuronal development, and MAP1B malfunction disturbs axonal growth. The MAP1 family proteins not only bind to microtubules but also interact with other cellular components. The MAP2/MAP4/tau superfamily also consists of three members, MAP2, MAP4, and tau. They share highly homologous microtubule-binding sequences in their microtubule-binding domains, while the other domains that protrude from the microtubule surfaces are totally distinct among the family members. MAP2 is mainly localized in cell bodies and dendrites, while tau is concentrated in axons. A low content of MAP4 is evenly distributed in cell bodies, dendrites and axons. Multiple variants of MAP2, MAP4, and tauare generated through alternative splicing, which is regulated in developmental stage- and tissue-specific manners. The MAP2/MAP4/tau superfamily proteins are highly active in forming, stabilizing, and stiffening microtubules. The minimal essential domain common among the family members was deduced to include the carboxyl terminal part of the Pro-rich region and a couple of microtubule-binding sequence repeats.

The role of the VQIVYK peptide in tau protein phosphorylation

Although it remains unclear whether they are related to one another, tau aggregation and phosphorylation are the main pathological hallmarks of the neuronal disorders known as tauopathies. The capacity to aggregate is impaired in a variant of the tau 3R isoform that lacks residues 306-311 (nomenclature for the largest CNS tau isoform) and hence, we have taken advantage of this feature to study how phosphorylation and aggregation may be related as well as the role of this six amino acid peptide (VQIVYK). Through these analyses, we found that the phosphorylation of the tau variant was higher than that of the complete tau protein and that not only the deletion of these residues, but also the interaction of these residues, in tau 3R, with thioflavin-S augmented tau phosphorylation by glycogen synthase kinase 3. In addition, the binding of the peptide containing the residues 306-311 to the whole tau protein provoked an increase in tau phosphorylation. This observation could be physiologically relevant as may suggest that tau-tau interactions, through those residues, facilitate tau phosphorylation. In summary, our data indicate that deletion of residues VQIVYK, in tau protein produces an increase in tau phosphorylation, without tau aggregation, because the VQIVYK peptide, that favors aggregation, is missing. On the other hand, when the whole tau protein interacts with thioflavin-S or the peptide VQIVYK, an increase in both aggregation and phosphorylation occurs.

In vitro tau fibrillization: Mapping protein regions

We have investigated the propensity to form fibrillar aggregates of a variety of fragments and variants of the tau protein under the influence of a tau fibrillization inducer: coenzyme Q 0. To better identify fibrillization hotspots, we compare the polymerization propensity of tau fragments containing the sequence of putative hotspots with that of tau variants with that same sequence deleted. We also investigate the effects of biologically occurring modifications such as phosphorylation and deamidation. We found that residues 305 to 335 are essential for in vitro tau fibrillization. Residues 306 to 311 facilitate in vitro assembly, but are not sufficient to mimic the in vivo fibrillization of tau. Furthermore, the propensity of the 306–311 sequence to form fibrils is highly decreased by chemical modifications of tyrosine 310 that are commonly found in vivo.

Survival Model Predictive Accuracy and ROC Curves

The predictive accuracy of a survival model can be summarized using extensions of the proportion of variation explained by the model, or R2, commonly used for continuous response models, or using extensions of sensitivity and specificity, which are commonly used for binary response models. In this article we propose new time-dependent accuracy summaries based on time-specific versions of sensitivity and specificity calculated over risk sets. We connect the accuracy summaries to a previously proposed global concordance measure, which is a variant of Kendall's tau. In addition, we show how standard Cox regression output can be used to obtain estimates of time-dependent sensitivity and specificity, and time-dependent receiver operating characteristic (ROC) curves. Semiparametric estimation methods appropriate for both proportional and nonproportional hazards data are introduced, evaluated in simulations, and illustrated using two familiar survival data sets.