Tau Phosphorylation Patterns Research Articles

NP-Collabs: Investigating Counterion-Mediated Bridges in the Multiply Phosphorylated Tau-R2 Repeat.

Tau is an intrinsically disordered (IDP) microtubule-associated protein (MAP) that plays a key part in microtubule assembly and organization. The function of tau can be regulated by multiple phosphorylation sites. These post-translational modifications are known to decrease the binding affinity of tau for microtubules, and abnormal tau phosphorylation patterns are involved in Alzheimer's disease. Using all-atom molecular dynamics simulations, we compared the conformational landscapes explored by the tau R2 repeat domain (which comprises a strong tubulin binding site) in its native state and with multiple phosphorylations on the S285, S289, and S293 residues, with four different standard force field (FF)/water model combinations. We find that the different parameters used for the phosphate groups (which can be more or less flexible) in these FFs and the specific interactions between bulk cations and water lead to the formation of a specific type of counterion bridge, termed nP-collab (for nphosphate collaboration, with n being an integer), where counterions form stable structures binding with two or three phosphate groups simultaneously. The resulting effect of nP-collabs on the tau-R2 conformational space differs when using sodium or potassium cations and is likely to impact the peptide overall dynamics and how this MAP interacts with tubulins. We also investigated the effect of phosphoresidue spacing and ionic concentration by modeling polyalanine peptides containing two phosphoserines located one-six residues apart. Three new metrics specifically tailored for IDPs (proteic Menger curvature, local curvature, and local flexibility) were introduced, which allow us to fully characterize the impact of nP-collabs on the dynamics of disordered peptides at the residue level.

Tau Phosphorylation Patterns in the Rat Cerebral Cortex After Traumatic Brain Injury and Sodium Selenate Effects: An Epibios4rx Project 2 Study.

Sodium selenate (SS) activates protein phosphatase 2 (PP2A) and reduces phosphorylated tau (pTAU) and late post-traumatic seizures after lateral fluid percussion injury (LFPI). In EpiBioS4Rx Project 2, a multi-center international study for post-traumatic targets, biomarkers, and treatments, we tested the target relevance and modification by SS of pTAU forms and PP2A and in the LFPI model, at two sites: Einstein and Melbourne. In Experiment 1, adult male rats were assigned to LFPI and sham (both sites) and naïve controls (Einstein). Motor function was monitored by neuroscores. Brains were studied with immunohistochemistry (IHC), Western blots (WBs), or PP2A activity assay, from 2 days to 8 weeks post-operatively. In Experiment 2, LFPI rats received SS for 7 days (SS0.33: 0.33 mg/kg/day; SS1: 1 mg/kg/day, subcutaneously) or vehicle (Veh) post-LFPI and pTAU, PR55 expression, or PP2A activity were studied at 2 days and 1 week (on treatment), or 2 weeks (1 week off treatment). Plasma selenium and SS levels were measured. In Experiment 1 IHC, LFPI rats had higher cortical pTAU-Ser202/Thr205-immunoreactivity (AT8-ir) and pTAU-Ser199/202-ir at 2 days, and pTAU-Thr231-ir (AT180-ir) at 2 days, 2 weeks, and 8 weeks, ipsilaterally to LFPI, than controls. LFPI-2d rats also had higher AT8/total-TAU5-ir in cortical extracts ipsilateral to the lesion (WB). PP2A (PR55-ir) showed time- and region-dependent changes in IHC, but not in WB. PP2A activity was lower in LFPI-1wk than in sham rats. In Experiment 2, SS did not affect neuroscores or cellular AT8-ir, AT180-ir, or PR55-ir in IHC. In WB, total cortical AT8/total-TAU-ir was lower in SS0.33 and SS1 LFPI rats than in Veh rats (2 days, 1 week); total cortical PR55-ir (WB) and PP2A activity were higher in SS1 than Veh rats (2 days). SS dose dependently increased plasma selenium and SS levels. Concordant across-sites data confirm time and pTAU form-specific cortical increases ipsilateral to LFPI. The discordant SS effects may either suggest SS-induced reduction in the numbers of cells with increased pTAU-ir, need for longer treatment, or the involvement of other mechanisms of action.

High-Fat Diet-Induced Diabetic Conditions Exacerbate Cognitive Impairment in a Mouse Model of Alzheimer's Disease Via a Specific Tau Phosphorylation Pattern.

Epidemiological evidence has demonstrated a clear association between diabetes mellitus and increased risk of Alzheimer's disease (AD). Cerebral accumulation of phosphorylated tau aggregates, a cardinal neuropathological feature of AD, is associated with neurodegeneration and cognitive decline. Clinical and experimental studies indicate that diabetes mellitus affects the development of tau pathology; however, the underlying molecular mechanisms remain unknown. In the present study, we used a unique diabetic AD mouse model to investigate the changes in tau phosphorylation patterns occurring in the diabetic brain. Tau-transgenic mice were fed a high-fat diet (n = 24) to model diabetes mellitus. These mice developed prominent obesity, severe insulin resistance, and mild hyperglycemia, which led to early-onset neurodegeneration and behavioral impairment associated with the accumulation of hyperphosphorylated tau aggregates. Comprehensive phosphoproteomic analysis revealed a unique tau phosphorylation signature in the brains of mice with diabetic AD. Bioinformatic analysis of the phosphoproteomics data revealed putative tau-related kinases and cell signaling pathways involved in the interaction between diabetes mellitus and AD. These findings offer potential novel targets that can be used to develop tau-based therapies and biomarkers for use in AD.

Mass spectrometric measurement of six site‐specific tau phosphorylations in CSF and blood of Alzheimer's disease patients

AbstractBackgroundHyperphosphorylation of tau is responsible for its loss of normal physiological function, gain of toxicity and its aggregation to form NFTs. Increased phosphorylated tau is detected in Alzheimer´s disease (AD) CSF and blood, and changes in the tau phosphorylation pattern may reflect different stages of the disease progression. A better understanding on how the levels of site‐specific phosphorylations are modulated across the AD continuum in these biofluids may help to provide with a more accurate diagnosis.MethodWe developed an LC‐MS method based on parallel reaction monitoring (PRM) using Orbitrap MS to measure tau phosphorylations at positions pT181, pS199, pT205, pT217 and pT231 in CSF and plasma, and additionally p396 in CSF. Tau enrichment prior to LC‐MS differed between biofluids. For CSF, 250 µl of sample were prepared by performing partial PCA protein precipitation, followed by SPE and tryptic digestion. For plasma, 1 ml samples were subjected to immunoprecipitation using a combination of several tau antibodies, followed by tryptic digestion and SPE.ResultThe performance of the method was first evaluated by analysis of CSF from neurochemically characterized AD patients and controls (n=40). All measured phosphorylations showed significant separation between groups (p<0.001) (Fig. 1). In addition, pT181, pT217 and pT231 showed good correlations with SIMOA immunoassays. In plasma, a set of AD patients and controls (n=26) were analysed. All phosphorylations showed an increase in AD, and most significantly pT205 (p=0.003) and pT231 (p=0.008) (Fig 2). Interestingly, these two phosphorylations were the ones that showed higher correlation with p‐tau CSF (Spearman r=0.52 and r=0.63, respectively). Plasma values were also compared to the respective SIMOA immunoassays. Finally, the method was used to measure samples from the Translational biomarker In Aging and Dementia (TRIAD) cohort (n=209), consisting of young individuals (<30 years old), cognitive unimpaired, mild cognitive impairment (MCI) and AD patients.ConclusionWith our LC‐MS method to quantify site‐specific p‐tau levels, we have established the tau phosphorylation pattern in AD biofluids. Our data indicates that phosphorylation at position T231 is the one that shows a higher fold change between AD and controls both in CSF and blood.

Functional protection in J20/VLW mice: a model of non-demented with Alzheimer's disease neuropathology.

Alzheimer's disease comprises amyloid-β and hyperphosphorylated Tau accumulation, imbalanced neuronal activity, aberrant oscillatory rhythms and cognitive deficits. Non-demented with Alzheimer's disease neuropathology defines a novel clinical entity with amyloid-β and Tau pathologies but preserved cognition. The mechanisms underlying such neuroprotection remain undetermined and animal models of non-demented with Alzheimer's disease neuropathology are currently unavailable. We demonstrate that J20/VLW mice (accumulating amyloid-β and hyperphosphorylated Tau) exhibit preserved hippocampal rhythmic activity and cognition, as opposed to J20 and VLW animals, which show significant alterations. Furthermore, we show that the overexpression of mutant human Tau in coexistence with amyloid-β accumulation renders a particular hyperphosphorylated Tau signature in hippocampal interneurons. The GABAergic septohippocampal pathway, responsible for hippocampal rhythmic activity, is preserved in J20/VLW mice, in contrast to single mutants. Our data highlight J20/VLW mice as a suitable animal model in which to explore the mechanisms driving cognitive preservation in non-demented with Alzheimer's disease neuropathology. Moreover, they suggest that a differential Tau phosphorylation pattern in hippocampal interneurons prevents the loss of GABAergic septohippocampal innervation and alterations in local field potentials, thereby avoiding cognitive deficits.

Tau phosphorylation and OPA1 proteolysis are unrelated events: Implications for Alzheimer's Disease

The neuropathological hallmarks of Alzheimer's Disease are plaques and neurofibrillary tangles. Yet, Alzheimer's is a complex disease with many contributing factors, such as energy-metabolic changes, which have been documented in autopsy brains from individuals with Alzheimer's and animal disease models alike. One conceivable explanation is that the interplay of age-related extracellular and intracellular alterations pertaining to Alzheimer's, such as cerebrovascular changes, protein aggregates and inflammation, evoke a mitochondrial response. However, it is not clear if and how mitochondria can contribute to Alzheimer's pathophysiology. This study focuses on one particular aspect of this question by investigating the functional interaction between the microtubule-associated protein tau and the mitochondrial inner membrane fusion machinery, which shows alterations in Alzheimer's brains. OPA1 is an essential inner membrane-fusion protein regulated by the two membrane proteases OMA1 and YME1L1. Assessment of OPA1 proteolysis—usually found in dividing mitochondria—and posttranslational tau modifications in mouse and human neuroblastoma cells under different experimental conditions clarified the relationship between these two pathways: OPA1 hydrolysis and phosphorylation or dephosphorylation of tau may coincide, but are not causally related. OPA1 cleavage did not alter tau's phosphorylation pattern. Conversely, tau's phosphorylation state did not induce nor correlate with OPA1 proteolysis. These results irrefutably demonstrate that there is no direct functional interaction between posttranslational tau modifications and the regulation of the OMA1-OPA1 pathway, which implies a common root cause modulating both pathways in Alzheimer's.

Tau in the gut, does it really matter?

The enteric nervous system plays a critical role in the regulation of gastrointestinal tract functions and is often referred to as the 'second brain' because it shares many features with the central nervous system. These similarities include among others a large panel of neurotransmitters, a large population of glial cells and a susceptibility to neurodegeneration. This close homology between the central and enteric nervous systems suggests that a disease process affecting the central nervous system could also involve its enteric counterpart. This was already documented in Parkinson's disease, the most common synucleinopathy, in which alpha-synuclein deposits are reported in the enteric nervous system in the vast majority of patients. Tau is another key protein involved in neurodegenerative disorders of the brain. Whether changes in tau also occur in the enteric nervous system during gut or brain disorders has just begun to be explored. The scope of the present article is therefore to review existing studies on the expression and phosphorylation pattern of tau in the enteric nervous system under physiological and pathological conditions and to discuss the possible occurrence of 'enteric tauopathies'.

The crystal structure of the catalytic domain of tau tubulin kinase 2 in complex with a small-molecule inhibitor.

Tau proteins play an important role in the proper assembly and function of neurons. Hyperphosphorylation of tau by kinases such as tau tubulin kinase (TTBK) has been hypothesized to cause the aggregation of tau and the formation of neurofibrillary tangles (NFTs) that lead to the destabilization of microtubules, thereby contributing to neurodegenerative diseases such as Alzheimer's disease (AD). There are two TTBK isoforms with highly homologous catalytic sites but with distinct tissue distributions, tau phosphorylation patterns and loss-of-function effects. Inhibition of TTBK1 reduces the levels of NFT formation involved in neurodegenerative diseases such as AD, whereas inhibition of TTBK2 may lead to the movement disorder spinocerebellar ataxia type 11 (SCA11). Hence, it is critical to obtain isoform-selective inhibitors. Structure-based drug design (SBDD) has been used to design highly potent and exquisitely selective inhibitors. While structures of TTBK1 have been reported in the literature, TTBK2 has evaded structural characterization. Here, the first crystal structure of the TTBK2 kinase domain is described. Furthermore, the crystal structure of human TTBK2 in complex with a small-molecule inhibitor has successfully been determined to elucidate the structural differences in protein conformations between the two TTBK isoforms that could aid in SBDD for the design of inhibitors that selectively target TTBK1 over TTBK2.

Small-molecule drug screening identifies drug Ro 31-8220 that reduces toxic phosphorylated tau in Drosophila melanogaster

The intraneuronal aggregates of hyperphosphorylated and misfolded tau (neurofibrillary tangles, NFTs) cause a stereotypical spatiotemporal Alzheimer's disease (AD) progression that correlates with the severity of the associated cognitive decline. Kinase activity contributes to the balance between neuron survival and cell death. Hyperactivation of kinases including the conventional protein kinase C (PKC) is a defective molecular event accompanying associative memory loss, tau phosphorylation, and progression of AD or related neurodegenerative diseases. Here, we investigated the ability of small therapeutic compounds (a custom library) to improve tau-induced rough-eye phenotype in a Drosophila melanogaster model of frontotemporal dementia. We also assessed the tau phosphorylation in vivo and selected hit compounds. Among the potential hits, we investigated Ro 31-8220, described earlier as a potent PKCα inhibitor. Ro 31-8220 robustly improved the rough-eye phenotype, reduced phosphorylated tau species in vitro and in vivo, reversed tau-induced memory impairment, and improved the fly motor functions. In a human neuroblastoma cell line, Ro 31-8220 reduced the PKC activity and the tau phosphorylation pattern, but we also have to acknowledge the compound's wide range of biological activity. Nevertheless, Ro 31-8220 is a novel therapeutic mitigator of tau-induced neurotoxocity.

Oxygen and Glucose Deprivation Alter Synaptic Distribution of Tau Protein: The Role of Phosphorylation.

Alterations in tau synaptic distribution are considered to underlie synaptic dysfunction observed in Alzheimer's disease (AD). In the present study, brain blood hypoperfusion was simulated in mouse brain slices, and tau levels and phosphorylation were investigated in total extracts, as well as in postsynaptic density fractions (PSDs) and non-PSDs obtained through differential extraction and centrifugation. Oxygen deprivation (OD) resulted in tau dephosphorylation at several AD-related residues and activation of GSK3β and phosphatase PP2A. On the contrary, glucose deprivation (GD) did not affect total levels of cellular tau or its phosphorylation despite inactivation of GSK3β. However, tau distribution in PSD and non-PSD fractions and the pattern of tau phosphorylation in these compartments is highly complex. In PSDs, tau was increased under GD conditions and decreased under OD conditions. GD resulted in tau dephosphorylation at Ser199, Ser262, and Ser396 while OD resulted in tau hyperphosphorylation at Ser199 and Ser404. In the non-PSD fraction, GD or OD resulted in lower levels of tau, but the phosphorylation status of tau was differentially affected. In GD conditions, tau was found dephosphorylated at Ser199, Thr205, and Ser404 and hyperphosphorylated at Ser262. However, in OD conditions tau was found hyperphosphorylated at Thr205, SerSer356, Ser396, and Ser404. Combined OD and GD resulted in degradation of cellular tau and dephosphorylation of PSD tau at Ser396 and Ser404. These results indicate that oxygen deprivation causes dephosphorylation of tau, while GD and OD differentially affect distribution of total tau and tau phosphorylation variants in neuronal compartments by activating different mechanisms.

Identification of the Tau phosphorylation pattern that drives its aggregation

Determining the functional relationship between Tau phosphorylation and aggregation has proven a challenge owing to the multiple potential phosphorylation sites and their clustering in the Tau sequence. We use here in vitro kinase assays combined with NMR spectroscopy as an analytical tool to generate well-characterized phosphorylated Tau samples and show that the combined phosphorylation at the Ser202/Thr205/Ser208 sites, together with absence of phosphorylation at the Ser262 site, yields a Tau sample that readily forms fibers, as observed by thioflavin T fluorescence and electron microscopy. On the basis of conformational analysis of synthetic phosphorylated peptides, we show that aggregation of the samples correlates with destabilization of the turn-like structure defined by phosphorylation of Ser202/Thr205.

Heterogeneous phosphorylation of microtubule-associated protein tau in cultured cells

Event Abstract Back to Event Heterogeneous phosphorylation of microtubule-associated protein tau in cultured cells Taeko Kimura1, Koichi Ishiguro2, Masato Hasegawa3 and Shin-ichi Hisanaga1* 1 Tokyo Metropolitan University, Molecular Neurosicence/Biological Sciences/Science and Engineering, Japan 2 Juntendo University, Neurology, Japan 3 Tokyo Metropolitan Institute of Medical Science, Dementia and Higher Brain Function, Japan Tau, a microtubule-associated protein, is a major component of aggregates seen in brains of tauopathies such as Alzheimer’s disease (AD) and frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). Tau is hyperphosphorylated in the aggregates and therefore the mechanism of hyperphosphorylation has been an intense research subject. Here, we applied the Phos-tag SDS-PAGE technique to characterize tau phosphorylation by cyclin-dependent kinase 5 (Cdk5) in vitro and in cultured cells. Cdk5-p25 phosphorylated tau at Ser404, Ser235, Thr205 and probably Se202 in that order. Among those four major in vitro Cdk5 phosphorylation sites, Ser404 was preferentially phosphorylated by Cdk5 in cultured cells, whereas Thr205 was not. Ser202 and Ser235 were phosphorylated by endogenous kinases. Tau existed in 12 different phosphorylation states in COS-7 cells with Ser202, Thr231 and Ser235 as major phosphorylation sites. p35-Cdk5 and p25-Cdk5 phosphorylated tau to a similar extent, when their expression levels were adjusted. Phosphorylation of FTDP-17 tau mutants was also analyzed by this method. While the mutants in the N-terminal or microtubule-binding region were phosphorylated identically as was wild type tau, the C-terminal mutants showed slightly different banding patterns from wild type tau. In particular, it was clear that R406W mutation resulted in loss of Ser404 phosphorylation. These results not only include novel and interesting information on tau phosphorylation but also indicate usefulness of Phos-tag SDS-PAGE in the analysis of site-specific and cellular phosphorylation pattern of tau. Keywords: Phosphorylation, Alzheimer’s disease, tau, in vitro, protein Conference: 14th Meeting of the Asian-Pacific Society for Neurochemistry, Kuala Lumpur, Malaysia, 27 Aug - 30 Aug, 2016. Presentation Type: Poster Presentation Session Topic: 14th Meeting of the Asian-Pacific Society for Neurochemistry Citation: Kimura T, Ishiguro K, Hasegawa M and Hisanaga S (2016). Heterogeneous phosphorylation of microtubule-associated protein tau in cultured cells. Conference Abstract: 14th Meeting of the Asian-Pacific Society for Neurochemistry. doi: 10.3389/conf.fncel.2016.36.00195 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 04 Aug 2016; Published Online: 11 Aug 2016. * Correspondence: Prof. Shin-ichi Hisanaga, Tokyo Metropolitan University, Molecular Neurosicence/Biological Sciences/Science and Engineering, Hachioji, Tokyo, Japan, hisanaga-shinichi@tmu.ac.jp Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Taeko Kimura Koichi Ishiguro Masato Hasegawa Shin-ichi Hisanaga Google Taeko Kimura Koichi Ishiguro Masato Hasegawa Shin-ichi Hisanaga Google Scholar Taeko Kimura Koichi Ishiguro Masato Hasegawa Shin-ichi Hisanaga PubMed Taeko Kimura Koichi Ishiguro Masato Hasegawa Shin-ichi Hisanaga Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Tamoxifen Inhibits CDK5 Kinase Activity by Interacting with p35/p25 and Modulates the Pattern of Tau Phosphorylation

Cyclin-dependent kinase 5 (CDK5) is a multifunctional enzyme that plays numerous roles, notably in brain development. CDK5 is activated through its association with the activators, p35 and p39, rather than by cyclins. Proteolytic procession of the N-terminal part of its activators has been linked to Alzheimer's disease and various other neuropathies. The interaction with the proteolytic product p25 prolongs CDK5 activation and modifies the substrate specificity. In order to discover small-molecule inhibitors of the interaction between CDK5 and p25, wehave used a bioluminescence resonance energy transfer (BRET)-based screening assay. Among the 1,760 compounds screened, the generic drug tamoxifen has been identified. The inhibition of the CDK5 activity by tamoxifen was notably validated by monitoring the phosphorylation state of tau protein. The study of the molecular mechanism of inhibition indicates that tamoxifen interacts with p25 to block the CDK5/p25 interaction and pave the way for new treatments of tauopathies.

Embryonic Stem Cell–Based Modeling of Tau Pathology in Human Neurons

Alterations in the microtubule (MT)-associated protein, tau, have emerged as a pivotal phenomenon in several neurodegenerative disorders, including frontotemporal dementia and Alzheimer's disease. Although compelling lines of evidence from various experimental models suggest that hyperphosphorylation and conformational changes of tau can cause its aggregation into filaments, the actual tau species and effective mechanisms that conspire to trigger the degeneration of human neurons remain obscure. Herein, we explored whether human embryonic stem cell-derived neural stem cells can be exploited to study consequences of an overexpression of 2N4R tau (two normal N-terminal and four MT-binding domains; n-tau) versus pseudohyperphosphorylated tau (p-tau) directly in human neurons. Given the involvement of tau in MT integrity and cellular homeostasis, we focused on the effects of both tau variants on subcellular transport and neuronal survival. By using inducible lentiviral overexpression, we show that p-tau, but not n-tau, readily leads to an MC-1-positive protein conformation and impaired mitochondrial transport. Although these alterations do not induce cell death under standard culture conditions, p-tau-expressing neurons cultured under non-redox-protected conditions undergo degeneration with formation of axonal varicosities sequestering transported proteins and progressive neuronal cell death. Our data support a causative link between the phosphorylation and conformational state of tau, microtubuli-based transport, and the vulnerability of human neurons to oxidative stress. They further depict human embryonic stem cell-derived neurons as a useful experimental model for studying tau-associated cellular alterations in an authentic human system.

Increased Tau Phosphorylation and Impaired Presynaptic Function in Hypertriglyceridemic ApoB-100 Transgenic Mice

AimsApoB-100 is the major protein component of cholesterol- and triglyceride-rich LDL and VLDL lipoproteins in the serum. Previously, we generated and partially described transgenic mice overexpressing the human ApoB-100 protein. Here, we further characterize this transgenic strain in order to reveal a possible link between hypeprlipidemia and neurodegeneration.Methods and ResultsWe analyzed the serum and cerebral lipid profiles, tau phosphorylation patterns, amyloid plaque-formation, neuronal apoptosis and synaptic plasticity of young (3 month old), adult (6 month old) and aging (10–11 month old) transgenic mice. We show that ApoB-100 transgenic animals present i) elevated serum and cerebral levels of triglycerides and ApoB-100, ii) increased cerebral tau phosphorylation at phosphosites Ser199, Ser199/202, Ser396 and Ser404. Furthermore, we demonstrate, that tau hyperphosphorylation is accompanied by impaired presynaptic function, long-term potentiation and widespread hippocampal neuronal apoptosis.ConclusionsThe results presented here indicate that elevated ApoB-100 level and the consequent chronic hypertriglyceridemia may lead to impaired neuronal function and neurodegeneration, possibly via hyperphosphorylation of tau protein. On account of their specific phenotype, ApoB-100 transgenic mice may be considered a versatile model of hyperlipidemia-induced age-related neurodegeneration.

Stable Mutated tau441 Transfected SH-SY5Y Cells as Screening Tool for Alzheimer’s Disease Drug Candidates

The role of hyperphosphorylation of the microtubule-associated protein tau in the pathological processes of several neurodegenerative diseases is becoming better understood. Consequently, development of new compounds capable of preventing tau hyperphosphorylation is an increasingly hot topic. For this reason, dependable in vitro and in vivo models that reflect tau hyperphosphorylation in human diseases are needed. In this study, we generated and validated an in vitro model appropriate to test potential curative and preventive compound effects on tau phosphorylation. For this purpose, a stably transfected SH-SY5Y cell line was constructed over-expressing mutant human tau441 (SH-SY5Y-TMHT441). Analyses of expression levels and tau phosphorylation status in untreated cells confirmed relevance to human diseases. Subsequently, the effect of different established kinase inhibitors on tau phosphorylation (e.g., residues Thr231, Thr181, and Ser396) was examined. It was shown with several methods including immunosorbent assays and mass spectrometry that the phosphorylation pattern of tau in SH-SY5Y-TMHT441 cells can be reliably modulated by these compounds, specifically targeting JNK, GSK-3, CDK1/5, and CK1. These four protein kinases are known to be involved in in vivo tau phosphorylation and are therefore authentic indicators for the suitability of this new cell culture model for tauopathies.

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.

Caffeine Modulates Tau Phosphorylation and Affects Akt Signaling in Postmitotic Neurons

Neuronal cell cycle reentry, which is associated with aberrant tau phosphorylation, is thought to be a mechanism of neurodegeneration in AD. Caffeine is a neuroprotective drug known to inhibit the cell cycle, suggesting that its neuroprotective nature may rely, at least in part, on preventing tau abnormalities secondary to its inhibitory effect on neuronal cell cycle-related pathways. Accordingly, we have explored in the present study the impact of caffeine on cell cycle-linked parameters and tau phosphorylation patterns in an attempt to identify molecular clues to its neuroprotective effect. We show that caffeine blocks the cell cycle at G1 phase in neuroblastoma cells and leads to a decrease in tau phosphorylation; similarly, exposure of postmitotic neurons to caffeine led to changes in tau phosphorylation concomitantly with downregulation of Akt signaling. Taken together, our results show a unique impact of caffeine on tau phosphorylation and warrant further investigation to address whether caffeine may help prevent neuronal death by preventing tau abnormalities secondary to aberrant entry into the cell cycle.

Phosphorylation differentiates tau-dependent neuronal toxicity and dysfunction

The heterogeneous pathology of tauopathies and the differential susceptibility of different neuronal types to WT (wild-type) and mutant tau suggest that phosphorylation at particular sites rather than hyperphosphorylation mediates toxicity or dysfunction in a cell-type-specific manner. Pan-neuronal accumulation of tau in the Drosophila CNS (central nervous system) specifically affected the MBs (mushroom body neurons), consistent with neuronal type-specific effects. The MB aberrations depended, at least in part, on occupation of two novel phosphorylation sites: Ser(238) and Thr(245). The degree of isoform-specific MB aberrations was paralleled by defects in associative learning, as blocking putative Ser(238) and Thr(245) phosphorylation yielded structurally normal, but profoundly dysfunctional, MBs, as animals accumulating the mutant protein exhibited strongly impaired associative learning. Similarly dysfunctional MBs were obtained by temporally restricting tau accumulation to the adult CNS, which also altered the tau phosphorylation pattern. Our data clearly distinguish tau-dependent neuronal degeneration and dysfunction and suggest that temporal differences in occupation of the same phosphorylation sites are likely to mediate these distinct effects of tau.

CSF phospho-tau is independent of age, cognitive status and gender of neurological patients

CSF phospho-tau (p-tau(181)) levels have shown good diagnostic utility in differential diagnosis of Alzheimer disease (AD). Unlike total-tau (t-tau), age related changes of this promising biomarker are sparsely studied. The aim of the study was to determine whether p-tau(181) is dependent on age, cognitive status or gender in patients with different neurological diseases who underwent diagnostic lumbar puncture and who had no clinical evidence of neurodegenerative diseases. CSF levels of p-tau(181) and total-tau (t-tau) of 46 neurologic patients (age range 22-89 years; 22 male, 24 female) were analyzed. Clinical diagnoses were cerebral ischaemia (n = 6), multiple sclerosis (n = 13), epileptic seizures (n = 3), polyneuropathy (n = 9) and other neurological diagnoses (n = 15). Cognitive performance was assessed by the German version of the CERAD battery. The mean level of p-tau(181) was in accordance with previous findings in neurological patients (42.8 +/- 15.3 pg/ml) and did not differ between neurological diseases. In contrast to t-tau (r = 0.38; P = 0.009), p-tau(181) did not correlate significantly to age (r = 0.15; P = 0.308). No influence of cognitive status or gender on p-tau(181) levels could be detected. The study corroborates the independence of p-tau(181) from age, cognitive status, gender and a wide spectrum of neurological diseases. The findings suggest that neither age related neurodegenerative processes nor ischaemic or inflammatory processes are accompanied by tau protein phosphorylation. In contrast, the data support the view that p-tau(181) seems to be a sign of the highly AD-specific pattern of tau phosphorylation during formation of neurofibrillary tangles.