Tau Phosphorylation Sites Research Articles

Predicting continuous amyloid PET values with CSF tau phosphorylation occupancies.

Cerebrospinal fluid (CSF) tau phosphorylation at multiple sites is associated with cortical amyloid and other pathologic changes in Alzheimer's disease. These relationships can be non-linear. We used an artificial neural network to assess the ability of 10 different CSF tau phosphorylation sites to predict continuous amyloid positron emission tomography (PET) values. CSF tau phosphorylation occupancies at 10 sites (including pT181/T181, pT217/T217, pT231/T231 and pT205/T205) were measured by mass spectrometry in 346 individuals (57 cognitively impaired, 289 cognitively unimpaired). We generated synthetic amyloid PET scans using biomarkers and evaluated their performance. Concentration of CSF pT217/T217 had low predictive error (average error: 13%), but also a low predictive range (ceiling 63 Centiloids). CSF pT231/T231 has slightly higher error (average error: 19%) but predicted through a greater range (87 Centiloids). Tradeoffs exist in biomarker selection. Some phosphorylation sites offer greater concordance with amyloid PET at lower levels, while others perform better over a greater range. Novel pTau isoforms can predict cortical amyloid burden. pT217/T217 accurately predicts cortical amyloid burden in low-amyloid individuals. Traditional CSF biomarkers correspond with higher levels of amyloid.

Quantitative profiling of posttranslational modifications of pathological tau via sarkosyl fractionation and mass spectrometry.

Tau protein aggregation is associated with posttranslational modifications (PTMs) in 75% of all dementia cases. The distribution of tau pathology and the presence of specific tau phosphorylation sites of interest are typically visualized and measured using antibodies. However, previous knowledge of the target epitopes is required. Additionally, antibodies can be used in a semi-quantitative manner but cannot be used to determine the absolute amount of tau or the extent of the modifications at specific sites or domains. Here we present a discovery assay that characterizes the global qualitative and quantitative tau modification landscape of a sample without a priori knowledge. Our workflow uses sarkosyl fractionation to extract the pathological tau species from sample-limited brain specimens, followed by mass spectrometry (MS) to characterize and quantify tau PTMs. The two-step MS-based proteomics approach includes an exploratory tau PTM analysis and a targeted full-length expressed stable isotope-labeled tau assay, which monitors specific unmodified tau peptides using a heavy isotope-labeled internal standard as a reference. This enables the absolute quantification of the respective tau peptides and the total tau amount in the sample, thus providing the modification extent of tau PTMs. This approach provides precise, comprehensive, qualitative and quantitative tau PTM profiling of the sample. It also enables the detailed molecular comparison of tau across multiple experiments, including a comparison between neurodegenerative diseases, stages of the disease, human patient heterogeneity and characterization of animal models. The approach is useful for studying the molecular features of pathological tau in neurodegeneration. The procedure requires 7-8 d and is suitable for users with expertise in targeted and untargeted MS-based protein analysis.

The AppNL‐G‐F/MAPT mouse model of Alzheimer’s Disease demonstrates increased levels of ptau217 in CSF, changes that correlate with increased cerebral p‐tau depositions

AbstractBackgroundCerebrospinal fluid (CSF) biomarkers reflecting the neuropathological hallmarks of Alzheimer’s disease (AD) have during the two last decades been developed and are commonly used to diagnose patients with AD patients. Previous studies have shown that CSF p‐tau217 particularly well can separate AD patients from healthy controls and that CSF levels of ptau217 correlate significantly with the amyloid burden and tau pathology in the brain (determined by PET). However, more studies are needed to fully understand the temporal relationship between CSF p‐tau217 changes and the accumulation tau in the brain. In this current study, we aim to investigate these temporal associations by the use of a novel knock‐in mouse model, AppNL‐G‐F expressing all six isoforms of human MAPT.MethodCerebrospinal fluid (CSF) and brain tissue will be collected from 3‐22 months old AppNL‐G‐F /MAPT and MAPT knock‐in mice. A pilot study of a subset of mice at different ages has been performed where levels of CSF ptau217 were analyzed using Mesoscale Discovery developed by Eli Lilly and Company. Mouse brain sections were immunostained against Aß and ptau181/217/231 to determine the cerebral p‐tau distribution.ResultIn CSF from our AppNL‐G‐F /MAPT double knock‐in (dKi) mouse model, we found age‐related increases in ptau217 levels compared to MAPT Ki mice. These changes in CSF ptau217 were associated with an increase of cerebral accumulation of p‐tau positive inclusions in the cortex and hippocampus of these mice. Additionally, we also found temporal differences in co‐localization between Aß plaques and the investigated phosphorylation sites of tau.ConclusionOur preliminary results from the AppNL‐G‐F/MAPT double knock‐in model, show early alterations in CSF ptau217 levels, changes that correlate in time with cerebral p‐tau positive accumulation as well as formation of Aß plaques. These findings indicate that alterations in CSF and cerebral p‐tau occurs early in the disease, when Aß pathology is widespread but no tau tangles are present. Furthermore, our preliminary data suggest temporal differences in phosphorylation sites of tau associated with Aß pathology. Additional CSF analysis and immunostainings will be performed before the AAIC conference.

Targeted Dephosphorylation of Tau by Phosphorylation Targeting Chimeras (PhosTACs) as a Therapeutic Modality.

Microtubule-associated protein tau is essential for microtubule assembly and stabilization. Hyperphosphorylation of the microtubule-associated protein tau plays an important pathological role in the development of Alzheimer's disease and other tauopathies. In vivo studies using kinase inhibitors suggest that reducing tau phosphorylation levels has therapeutic potential; however, such approaches showed limited benefits. We sought to further develop our phosphorylation targeting chimera (PhosTAC) technology to specifically induce tau dephosphorylation. Herein, we use small molecule-based PhosTACs to recruit tau to PP2A, a native tau phosphatase. PhosTACs induced the formation of a stable ternary complex, leading to rapid, efficient, and sustained tau dephosphorylation, which also correlated with the enhanced downregulation of tau protein. Mass spectrometry data validated that PhosTACs downregulated multiple phosphorylation sites of tau. We believe that PhosTAC possesses several advantages over current strategies to modulate tau phosphorylation and represents a new avenue for disease-modifying therapies for tauopathies.

Tau immunotherapy in Alzheimer's disease and progressive supranuclear palsy.

Tubulin-associated unit (tau) has been associated with more than 25 neurological disorders-the so-called tauopathies. Hence, finding a novel therapeutic agent targeting tau to halt the progression of diseases has been of interest. Alzheimer's disease (AD) and progressive supranuclear palsy (PSP) are the most studied tauopathies. AD is characterized by two cardinal pathological mechanisms: amyloid β (Aβ) plaques and neurofibrillary tangles (NFTs), leading to atrophy of the brain. Over the last few years, attention has been on NFTs composed of the hyperphosphorylated microtubule-associated protein tau. Tau contributes to the synaptic plasticity of axons; hyperphosphorylated and aggregated tau have been shown to increase the likelihood of cognitive impairments. PSP is also associated with tau accumulation in NFTs and neuropil threads, making this condition a candidate for tau-targeted therapies. Many tau-targeting therapies have been developed, and clinical trials are being conducted. Tau-targeting drugs are classified into four subgroups based on the pathological target: tau phosphorylation inhibitors, stabilizers of microtubules, enhancing tau clearance, and tau aggregation inhibitors. On the other hand, the desired specificity and sensitivity of tau immunotherapy agents without interrupting normal proteome are the fundamental point of tremendous attention. Starting with animal studies of these therapies to clinical trials, both are divided into passive and active immunotherapies, while passive immunotherapies are the method of desire. Targeting aggregation and phosphorylation sites of tau is the chief target of therapies. This article reviews the latest animal and clinical studies of tau-based immunotherapies and tau-targeted drugs for AD and PSP.

MultiTEP‐based conjugated vaccines targeting various phosphorylated tau epitopes are immunogenic and effective in the PS19 mouse model

AbstractBackgroundPathological tau correlates well with cognitive impairments in Alzheimer’s disease (AD) patients and represents a promising immunotherapy target. In theory, targeting an appropriate B‐cell epitope in pathological tau could effectively reduce the pathology without affecting normal native tau. Hyperphosphorylation of tau is a well‐known feature of Alzheimer’s and other tauopathies, with many disease‐specific phosphorylation sites documented. Accordingly, we attached several disease‐related phosphorylated tau peptides to our universal vaccine platform, MultiTEP that is safe and extremely immunogenic in various disease models of AD, rabbits, and aged monkeys. We hypothesized that high immunogenicity of MultiTEP‐based vaccines, coupled with the selectivity of the immune responses, will allow the identification of pathological and protective phosphorylation sites, thus leading to the generation of vaccines, which would preferentially clear the toxic forms of tau without disrupting the normal brain homeostasis.MethodA genetically engineered variant of MultiTEP was generated to enable chemoselective bioconjugation of synthetic peptides under mild aqueous conditions. MultiTEP‐based vaccines targeting p‐tau 202/205, p‐tau 396/404, p‐tau 422 were tested in the PS19 mouse model of AD and other tauopathies. Antibody specificity and titers were tested via ELISA. Brain total and phosphorylated soluble and insoluble tau were measured by ELISA and IHC.ResultAll three vaccines generated robust and selective immune responses specific to phosphorylated tau in mice. The generated antibodies selectively bound to the appropriate phosphorylated, but not non‐phosphorylated form of the same peptide. Importantly, these antibodies effectively reduce/cleared pathological tau in the brains of immunized mouse model of tauopathy. The MultiTEP immunogenicity was sufficient to generate robust immune responses after two immunizations, even without adjuvant.ConclusionMultiTEP‐based conjugated vaccines could be used to generate robust immune responses selective to multiple phosphorylation sites of tau. This, in conjunction with a previous report on MultiTEP‐based pE3Aβ vaccine, suggests that the methodology is universally applicable for various post‐translational modifications. Our technology can facilitate the identification of optimal therapeutic targets of p‐tau for generating an active immunotherapeutic strategy that can be used in conjunction with pE3Aβ vaccine. If successful, preventive vaccinations targeting disease‐related tau and/or Aβ molecules could at least delay AD and related neurodegenerative disorders.

Plasma ratio of phospho‐ to non‐phospho‐tau at threonine 217 identifies brain amyloid burden as measured by quantitative amyloid PET in the PARIS sub‐study of IDEAS

AbstractBackgroundAccumulation of amyloid beta plaques and tau tangles in the brain are sequential pathological hallmarks of Alzheimer’s disease. Specific measurement of tau phosphorylation sites in plasma samples can be used to investigate the presence of both amyloid plaque and tau tangle brain pathology.MethodA tandem mass spectrometry assay that quantifies phosphorylation of tau at threonine residues 181 and 217 in plasma was developed. Both phosphorylated (phospho) and non‐phosphorylated (non‐phospho) peptides containing tau residues 181 and 217 as well as the ratio of phospho to non‐phospho at each of these phosphorylation sites can be calculated from a blood sample. We tested the concordance between these measures and brain amyloid burden in PARIS study participants, an IDEAS sub‐study in patients with mild cognitive impairment or dementia. K2EDTA plasma samples from PARIS participants (n=221) were analyzed for tau phosphorylation and Aβ42/40.ResultsThe amount of phosphorylation at both 181 and 217 sited were significantly higher in patients who were amyloid PET positive, while PET negative patients showed very low amounts of phosphorylation at 217 (1.2 pg/mL vs. 4.9 pg/mL; p <2e‐16). The ratio of phospho‐tau to non‐phospho‐tau 217 showed high correlation (Spearman’s rank correlation r=0.78; p <2e‐16) with quantitative amyloid PET, demonstrating that phosphorylation of tau on threonine‐217 can quantitatively identify the amount of amyloid burden. This ratio of phospho‐tau to non‐phospho‐tau also correlated better with amyloid status than the concentration of phospho‐tau alone, with area under the curve (AUC) for detecting brain amyloid burden improving from 0.74 (95%CI 0.67‐0.82) to 0.81 (95%CI 0.75‐0.88) for 181 and from 0.92 (95%CI 0.88‐0.96) to 0.95 (95%CI 0.92‐0.98) for 217. Combining the phospho‐tau‐217 with the Aβ42/40 ratios for the same sample further increased the AUC to 0.96 (95%CI 0.93‐0.99).ConclusionThis novel mass spectrometry‐based quantitative assay for plasma phospho‐tau and non‐phospho‐tau species has excellent analytical and clinical validity performance characteristics. Combining the ratio of tau phosphorylation at 217 with plasma Aβ42/40 can help identify brain amyloid pathology with a performance that rivals that of CSF or PET tests.

Site-Specific Phospho-Tau Aggregation-Based Biomarker Discovery for AD Diagnosis and Differentiation.

Tau aggregates are present in multiple neurodegenerative diseases known as "tauopathies," including Alzheimer's disease (AD), Pick's disease (PiD), progressive supranuclear palsy (PSP), and corticobasal degeneration (CBD). Such misfolded tau aggregates are therefore potential sources for tauopathy biomarker discovery. Using the tau antibody screening approach targeting high-molecular-weight misfolded tau aggregates, we tested several tau antibodies and a comprehensive set of site-specific phospho-tau (p-tau) antibodies targeting tau phosphorylation sites showing high frequencies in AD subjects. Our screens revealed that site-specific p-tau antibodies can not only differentiate AD from non-AD brains, but also discriminate AD from rare tauopathies PiD, PSP, and CBD brains. Differential detection of tau aggregates identified several novel p-tau sites as potential new biomarkers. As a proof-of-principle example, we showed that p-tau198 is a novel promising AD biomarker with sensitivity and specificity comparable with the existing biomarkers p-tau181 and p-tau217. Our results demonstrated that p-tau198 detection can not only differentiate AD from non-AD controls, but also diagnose AD from related 4R tauopathies PSP and CBD with AUCs of 0.96-0.99 (95% CI ranges from 0.90 to 1.00). Promisingly, p-tau198 was able to discriminate mild cognitive impairment from cognitively normal brains with an AUC of 0.75 (95% CI = 0.58-0.92). Our work provides a new avenue for developing diagnosis and differentiation tools for AD and related tauopathies.

MicroRNA-140-5p inhibitor attenuates memory impairment induced by amyloid-ß oligomer in vivo possibly through Pin1 regulation.

The peptidyl-prolyl cis/trans isomerase, Pin1, has a protective role in age-related neurodegeneration by targeting different phosphorylation sites of tau and the key proteins required to produce Amyloid-β, which are the well-known molecular signatures of Alzheimer's disease (AD) neuropathology. The direct interaction of miR-140-5p with Pin1 mRNA and its inhibitory role in protein translation has been identified. The main purpose of this study was to investigate the role of miRNA-140-5p inhibition in promoting Pin1 expression and the therapeutic potential of the AntimiR-140-5p in the Aß oligomer (AßO)-induced AD rat model. Spatial learning and memory were assessed in the Morris water maze. RT-PCR, western blot, and histological assays were performed on hippocampal samples at various time points after treatments. miRNA-140-5p inhibition enhanced Pin1 and ADAM10 mRNA expressions but has little effect on Pin1 protein level. The miRNA-140-5p inhibitor markedly ameliorated spatial learning and memory deficits induced by AßO, and concomitantly suppressed the mRNA expression of inflammatory mediators TNFα and IL-1β, and phosphorylation of tau at three key sites (thr231, ser396, and ser404) as well as increased phosphorylated Ser473-Akt. According to our results, Antimir-140-mediated improvement of AβO-induced neuronal injury and memory impairment in rats may provide an appropriate rationale for evaluating miR-140-5p inhibitors as a promising agent for the treatment of AD.

Human genome-wide analysis and identification of the hyperphosphorylation-elicited interactions between subarachnoid tau protein and phosphoprotein-binding domains.

Abnormally hyperphosphorylated tau can be recognized by a variety of phosphoprotein-binding domains (PBDs) to elicit downstream tau signaling in neuropathology, which has been found to have a potential association with subarachnoid hemorrhage. In this study, the genome-wide binding behavior of tau phosphorylation sites (p-sites) to PBDs involved in subarachnoid hyperphosphorylation events was systematically profiled at molecular level by integrating peptide docking, structural minimization, affinity scoring, and binding assay, from which a number of potent PBD-p-site interaction pairs were identified. It was revealed that the PBD domains exhibit distinct binding preferences for phosphotyrosine, phosphoserine, and phosphothreonine p-sites; the PBD-recognition specificity of different tau p-sites is not overlapped with each other, and their phosphorylations would therefore regulate varying biological functions in tau signaling. A number of PBD-p-site pairs were identified to have potent binding potency as compared to others. The KCIP-pS[393-399] pair was found as a strong binder, which was further optimized with a rational peptide design protocol to derive a number of affinity-improved phosphopeptides. Structural analysis revealed diverse noncovalent chemical forces across the complex interface of KCIP domain with a designed high-affinity pS[393-399]-d4, which confers both stability and specificity to the domain-peptide complex system, with affinity improved by 10.9-fold relative to the native pS[393-399].

Tau phosphorylation sites serine202 and serine396 are differently altered in chronic traumatic encephalopathy and Alzheimer's disease.

Chronic traumatic encephalopathy (CTE) is a neurodegenerative tauopathy associated with repetitive head impacts (RHI) typically sustained by contact sport athletes. Post-translation modifications to tau in CTE have not been well delineated or compared to Alzheimer's disease (AD). We measured phosphorylated tau epitopes within dorsolateral frontal cortex from post mortem brains with neither CTE nor AD (n = 108), CTE (n = 109), AD (n = 223), and both CTE and AD (n = 33). Levels of hyperphosphorylated tau (p-tau)202 , p-tau231 , and p-tau396 were significantly increased in CTE. Total years of RHI exposure was significantly associated with increased p-tau202 levels (P = .001), but not p-tau396 . Instead, p-tau396 was most closely related to amyloid beta (Aβ)1-42 levels (P < .001). The p-tau202 :p-tau396 ratio was significantly increased in early and late CTE compared to AD. In frontal cortex, p-tau202 is the most upregulated p-tau species in CTE, while p-tau396 is most increased in AD. p-tau202 and p-tau396 measurements may aid in developing biomarkers for disease.

Characterizing neurofibrillary tangle maturity of phosphorylated tau fluid biomarkers in Alzheimer's disease

Phosphorylated tau (p-tau) cerebrospinal fluid and plasma biomarkers have become more accurate in predicting Alzheimer's disease (AD) pathology. Although p-tau fluid biomarkers have been studied in vivo for nearly 20 years, tau phosphorylation sites have not been systematically studied in the context of neurofibrillary tangle maturity in the postmortem brain. Neurofibrillary tangles have a lifespan that is defined by three maturity levels: pretangles, mature tangles, and ghost tangles. Our goal was to characterize four tau phosphorylation sites (pT181, pT205, pT217, and pT231) in postmortem brain to determine which neurofibrillary tangle maturity level they preferentially recognize.The FLorida Autopsied Multi-Ethnic (FLAME) cohort was queried for cases at each Braak stage (I-VI). We excluded α-synucleinopathies, TDP-43 proteinopathies, and non-AD primary tauopathies, and those lacking available tissue. Ethnoracial diversity was prioritized for final case selection. A total of 24 cases were evaluated with each Braak stage containing 2 males and 2 females. For Braak stages IV-VI, only cases with a neuropathologic diagnosis of typical AD were selected. Posterior hippocampus was stained by immunohistochemistry for four antibodies: AT270 (pT181), pT205, pT217, and AT180 (pT231). Slides were digitized using Leica's Aperio technology to enable quantification of tau burden in the CA1 subsector and subiculum of the hippocampus.All tau phosphorylation sites recognized pretangles and mature tangles, and rarely recognized ghost tangles. Digital pathology measures of pT181, pT205, pT217, and pT231 burden showed subtle increases from Braak I-III. A greater increase was observed from Braak IV onward, except in pT217 where the burden decreased from Braak IV to Braak V before maxing out in Braak VI. pT205 and pT217 had the largest increase between Braak III and Braak VI compared to pT181 and pT231.pT181, pT205, pT217, and pT231 recognize primarily early neurofibrillary tangle maturity levels. This finding could explain why these p-tau sites are recognized in fluids before symptom onset. Future studies will compare these p-tau sites to other commonly used tau antibodies for AD research, including AT8 and PHF-1, as well as to in vivo measures in autopsied study participants who underwent antemortem biomarker evaluation.

G protein-coupled receptor kinases are associated with Alzheimer's disease pathology.

Alzheimer's disease (AD) is characterised by extracellular deposition of amyloid-β (Aβ) in amyloid plaques and intracellular aggregation and accumulation of hyperphosphorylated tau in neurofibrillary tangles (NFTs). Although several kinases have been identified to contribute to the pathological phosphorylation of tau, kinase-targeted therapies for AD have not been successful in clinical trials. Critically, the kinases responsible for numerous identified tau phosphorylation sites remain unknown. G protein-coupled receptor (GPCR) kinases (GRKs) have recently been implicated in phosphorylation of non-GPCR substrates, for example, tubulin and α-synuclein, and in neurological disorders, including schizophrenia and Parkinson's disease. Accordingly, we investigated the involvement of GRKs in the pathophysiology of AD. We performed a comprehensive immunohistochemical and biochemical analysis of the ubiquitously expressed GRKs, namely, GRK2, 3, 5 and 6, in postmortem human brain tissue of control subjects and AD patients. GRKs display unique cell-type-specific expression patterns in neurons, astrocytes and microglia. Levels of GRKs 2, 5 and 6 are specifically decreased in the CA1 region of the AD hippocampus. Biochemical evidence indicates that the GRKs differentially associate with total, soluble and insoluble pools of tau in the AD brain. Complementary immunohistochemical studies indicate that the GRKs differentially colocalise with total tau, phosphorylated tau and NFTs. Notably, GRKs 3 and 5 also colocalise with amyloid plaques. These studies establish a link between GRKs and the pathological phosphorylation and accumulation of tau and amyloid pathology in AD brains and suggest a novel role for these kinases in regulation of the pathological hallmarks of AD.

Cerebrospinal fluid tau biomarkers in the prediction and concordance of neurofibrillary tangle and amyloid pathology

AbstractBackgroundCerebrospinal fluid (CSF) measures of tau (p‐tau181 and t‐tau) are central to the research diagnostic criteria for Alzheimer’s disease (AD). Nevertheless, alternative tau phosphorylation sites or tau fragments merit further evaluation to better index neurofibrillary tangle (NFT) and amyloid‐β (Aβ) pathology.MethodWe evaluated 168 participants from the TRIAD cohort (young controls, cognitively normal controls (CN), mild cognitive impairment, frontotemporal dementia and AD patients). LUMIPULSE® G1200 (Fujirebio) was used to measure CSF p‐tau181 and t‐tau. CSF p‐tau231 was quantified using a prototype ELISA assay (ADx NeuroSciences). CSF p‐tau217 and tau368 was measured using an in‐house Simoa assay (University of Gothenburg). For PET ([18F]MK6240 and [18F]AZD4694), standard uptake value ratios (SUVR) were determined using the cerebellar and cerebellar cortex as reference tissue, respectively. Finally, ROC curves were generated to investigate the predictive power of the CSF biomarkers.ResultAll CSF p‐tau biomarkers equally associated with [18F]MK6240 (r&gt;0.51), while CSF p‐tau231 correlated more to [18F]AZD4694 (r=0.815) than other tau biomarkers. ROC curves revealed p‐tau biomarkers accurately detected Braak I‐II (AUC&gt;0.905) and III‐IV positivity (AUC&gt;0.940), with p‐tau217 significantly out performing p‐tau231 and p‐tau181 at both stages. Tau368/t‐tau ratio was the superior biomarker in the prediction of Braak V‐VI positivity. P‐tau217 and p‐tau231 demonstrated high accuracy in predicting Aβ positivity (ROC&gt;0.950). This was significantly better than p‐tau181 (p‐tau217, P&lt;0.01; p‐tau231, P&lt;0.001). Prediction of Aβ positivity was also high at the pre‐symptomatic stage for p‐tau231 (ROC&gt;0.9) and again was significantly superior to p‐tau181 (P&lt;0.01). All p‐tau biomarkers demonstrated full concordance (CSF‐/PET‐) with [18F]MK6240 and [18F]AZD4694 for young controls and FTD subjects. P‐tau217 and p‐tau231 demonstrated high concordance with [18F]MK6240 and discordant cases were restricted to the CSF+/PET‐ quadrant (Figure 1). In the same manner, CSF p‐tau217 and p‐tau231 demonstrated the highest concordance with [18F]AZD4694, although subjects were observed in both discordant quadrants (Figure 2).ConclusionAll CSF p‐tau biomarkers relate to NFT pathology to the same degree, however, tau368/t‐tau seemingly characterises NFT pathology at an advanced stage. CSF p‐tau217 and p‐tau231 are better predictors of Aβ pathology, with p‐tau231 a stronger predictor of Aβ pathology at the pre‐symptomatic stage.

Functional Interactions of Tau Phosphorylation Sites That Mediate Toxicity and Deficient Learning in Drosophila melanogaster.

Hyperphosphorylated Tau protein is the main component of the neurofibrillary tangles, characterizing degenerating neurons in Alzheimer’s disease and other Tauopathies. Expression of human Tau protein in Drosophila CNS results in increased toxicity, premature mortality and learning and memory deficits. Herein we use novel transgenic lines to investigate the contribution of specific phosphorylation sites previously implicated in Tau toxicity. These three different sites, Ser238, Thr245, and Ser262 were tested either by blocking their phosphorylation, by Ser/Thr to Ala substitution, or pseudophosphorylation, by changing Ser/Thr to Glu. We validate the hypothesis that phosphorylation at Ser262 is necessary for Tau-dependent learning deficits and a “facilitatory gatekeeper” to Ser238 occupation, which is linked to Tau toxicity. Importantly we reveal that phosphorylation at Thr245 acts as a “suppressive gatekeeper”, preventing phosphorylation of many sites including Ser262 and consequently of Ser238. Therefore, we elucidate novel interactions among phosphosites central to Tau mediated neuronal dysfunction and toxicity, likely driven by phosphorylation-dependent conformational plasticity.

GSKIP-Mediated Anchoring Increases Phosphorylation of Tau by PKA but Not by GSK3beta via cAMP/PKA/GSKIP/GSK3/Tau Axis Signaling in Cerebrospinal Fluid and iPS Cells in Alzheimer Disease.

Based on the protein kinase A (PKA)/GSK3β interaction protein (GSKIP)/glycogen synthase kinase 3β (GSK3β) axis, we hypothesized that these might play a role in Tau phosphorylation. Here, we report that the phosphorylation of Tau Ser409 in SHSY5Y cells was increased by overexpression of GSKIP WT more than by PKA- and GSK3β-binding defective mutants (V41/L45 and L130, respectively). We conducted in vitro assays of various kinase combinations to show that a combination of GSK3β with PKA but not Ca2+/calmodulin-dependent protein kinase II (CaMK II) might provide a conformational shelter to harbor Tau Ser409. Cerebrospinal fluid (CSF) was evaluated to extend the clinical significance of Tau phosphorylation status in Alzheimer’s disease (AD), neurological disorders (NAD), and mild cognitive impairment (MCI). We found higher levels of different PKA–Tau phosphorylation sites (Ser214, Ser262, and Ser409) in AD than in NAD, MCI, and normal groups. Moreover, we used the CRISPR/Cas9 system to produce amyloid precursor protein (APPWT/D678H) isogenic mutants. These results demonstrated an enhanced level of phosphorylation by PKA but not by the control. This study is the first to demonstrate a transient increase in phosphor-Tau caused by PKA, but not GSK3β, in the CSF and induced pluripotent stem cells (iPSCs) of AD, implying that both GSKIP and GSK3β function as anchoring proteins to strengthen the cAMP/PKA/Tau axis signaling during AD pathogenesis.

Tau Phosphorylation Rates Measured by Mass Spectrometry Differ in the Intracellular Brain vs. Extracellular Cerebrospinal Fluid Compartments and Are Differentially Affected by Alzheimer's Disease.

Tau protein aggregation into neurofibrillary tangles in the central nervous system contributes to the etiology of certain neurodegenerative disorders, including Alzheimer’s disease (AD). Though the mechanism of tau destabilization is not fully understood yet, tau protein has been found to be hyperphosphorylated in tau aggregates. To investigate this further, we developed a highly sensitive and specific mass spectrometry (MS) method using parallel reaction monitoring (PRM) to identify tau phosphorylation sites. This method enables us to compare the abundance of phosphorylation sites in tau proteins in the brain and cerebrospinal fluid (CSF) in humans with and without AD. We detected 29 distinct phosphorylated tau (p-tau) sites in full-length tau from soluble human brain lysate and 12 sites on truncated tau in CSF, mainly in the mid-domain. Brain soluble tau phosphorylation sites are localized on three domains including a proline-rich mid-domain, the C-terminus, and a cluster on the N-terminal projection domain not previously characterized. Some phosphorylation sites increased in CSF, while others decreased compared to brain. Notably, phosphorylation on T205 and S208, recognized by AT8 antibody defining Braak stages of brain tau aggregation, were not detected in normal brain soluble tau but were found in the CSF. Comparison of the p-tau rates from the brain and the CSF indicated that the abundance of phosphorylated sites varied in a site-specific manner. CSF tau proteins from non-AD participants were significantly hyperphosphorylated on T111, T205, S208, T217 and T231. In AD CSF, hyperphosphorylation on these sites was exacerbated, and phosphorylation on T153 and T175 specifically were detected. This supports the hypothesis that tau hyperphosphorylation could be a physiological process amplified by AD pathology. Conversely, we found that S202 was hypophosphorylated in CSF and was not hyperphosphorylated in AD, demonstrating that p-tau isoforms could have different metabolisms depending on which sites are phosphorylated. These site-specific p-tau rates are independent of tau concentration and distinct of current CSF tau and p-tau assays measuring tau isoforms levels. Targeted MS multiplexing ability and high-throughput capacity lets us envision the use of these new p-tau measurements as promising biomarkers for AD diagnosis and tracking therapeutic responses.

Platelet Tau Protein as a Potential Peripheral Biomarker in Alzheimer’s Disease: An Explorative Study

Cerebrospinal fluid (CSF) measures of tau and amyloid proteins have now been largely accepted to be a diagnostic tool to aid the clinical diagnosis of Alzheimer's disease (AD), but CSF is not routinely obtained in most clinical settings. There is a need, therefore, to uncover additional readily accessible peripheral biomarkers that will enable comprehensive detection of AD-specific proteins in blood and blood derivates. Blood platelets contain proteins found in neuronal cell lines, including tau protein. Since tau protein is a characteristic of AD-neuropathology, platelet tau protein may be closely related to the central nervous process occurring in neurodegeneration. Platelets from 25 AD and 26 control subjects were analysed for the microtubule-binding and C-terminal region, as well as two tau phosphorylation sites (Ser202/Thr205 and Thr181). Tau protein measures did not discriminate between AD and control individuals. However, subjects with MMSE 24-27 had elevated C-terminal end tau protein (p=0.049) compared to those with MMSE >27, whereas older AD subjects (>80 years) showed higher t-tau protein in comparison to younger AD (<80 years; p=0.009) and control (<80 years; p=0.011) participants. These initial findings not only confirm that platelet tau protein can be measured, but also indicate that platelet tau measures merit further study as they may be useful in indicating early stages of cognitive impairment. Further studies on larger number of participants are needed to confirm our findings.

Tau Antibody Structure Reveals a Molecular Switch Defining a Pathological Conformation of the Tau Protein

Tau antibodies have shown therapeutic potential for Alzheimer’s disease and several are in clinical trials. As a microtubule-associated protein, tau relies on dynamic phosphorylation for its normal functions. In tauopathies, it becomes hyperphosphorylated and aggregates into toxic assemblies, which collectively lead to neurodegeneration. Of the phospho-epitopes, the region around Ser396 has received particular attention because of its prominence and stability in tauopathies. Here we report the first structure of a monoclonal tau antibody in complex with the pathologically important phospho-Ser396 residue. Its binding region reveals tau residues Tyr394 to phospho-Ser396 stabilized in a β-strand conformation that is coordinated by a phospho-specific antigen binding site. These details highlight a molecular switch that defines this prominent conformation of tau and ways to target it. Overall, the structure of the antibody-antigen complex clarifies why certain phosphorylation sites in tau are more closely linked to neurodegeneration than others.

Left-handed polyproline-II helix revisited: proteins causing proteopathies

Left-handed polyproline-II type helix is a regular conformation of polypeptide chain not only of fibrous, but also of folded and natively unfolded proteins and peptides. It is the only class of regular secondary structure substantially represented in non-fibrous proteins and peptides on a par with right-handed alpha-helix and beta-structure. In this study, we have shown that polyproline-II helix is abundant in several peptides and proteins involved in proteopathies, the amyloid-beta peptides, protein tau and prion protein. Polyproline-II helices form two interaction sites in the amyloid-beta peptides, which are pivotal for pathogenesis of Alzheimer’s disease (AD). It also with high probability is the structure of the majority of tau phosphorylation sites, important for tau hyperphosphorylation and formation of neurofibrillary tangles, a hallmark of AD. Polyproline-II helices form large parts of the structure of the folded domain of prion protein. They can undergo conversion to beta-structure as a result of relatively small change of one torsional angle of polypeptide chain. We hypothesize that in prions and amyloids, in general polyproline-II helices can serve as structural elements of the normal structure as well as dormant nuclei of structure conversion, and thus play important role in structure changes leading to the formation of fibrils.