Formation Of Paired Helical Filaments Research Articles

Nutrients and polyphenols-rich Sorghum bicolor genotypes as complementary therapy for Alzheimer’s disease

AbstractAlzheimer’s disease (AD) is a progressive neurodegenerative disorder and most common cause of dementia among older people. The main pathological hallmarks of AD are formation of insoluble amyloid beta senile plaques and paired helical filaments of neurofibrillary tangles. AD features gradual memory decline, mild to severe cognitive impairment, eventually total dependence of patients on caregivers. Currently available drugs have not been able to modify AD pathology. This has drawn increasing attention to plant food materials with high nutritional and bioactive constituents as potential complementary therapy for AD. Sorghum bicolor is a widely available cost-effective source of proteins, fats, crude fibres, biopeptides and polyphenols which are vital for human wellbeing. This review discussed the major mechanisms underlying AD pathology. The nutritional and bioactive constituents of Sorghum bicolor grains were extensively described. There is limited report on anti-AD activities of sorghum grains. Therefore, the pharmacological mechanisms of action including scavenging of reactive oxygen species, inhibition of oxidative stress, anti-acetylcholinesterase activity and modulation of mitophagy were only speculated. This comprehensive update suggests more robust innovative studies that will provide critical theoretical details necessary to promote utilization of sorghum grains as functional food or source of bioactive molecules for AD therapy.

Role of Cytoskeletal Elements in Regulation of Synaptic Functions: Implications Toward Alzheimer's Disease and Phytochemicals-Based Interventions.

Alzheimer's disease (AD), a multifactorial disease, is characterized by the accumulation of neurofibrillary tangles (NFTs) and amyloid beta (Aβ) plaques. AD is triggered via several factors like alteration in cytoskeletal proteins, a mutation in presenilin 1 (PSEN1), presenilin 2 (PSEN2), amyloid precursor protein (APP), and post-translational modifications (PTMs) in the cytoskeletal elements. Owing to the major structural and functional role of cytoskeletal elements, like the organization of axon initial segmentation, dendritic spines, synaptic regulation, and delivery of cargo at the synapse; modulation of these elements plays an important role in AD pathogenesis; like Tau is a microtubule-associated protein that stabilizes the microtubules, and it also causes inhibition of nucleo-cytoplasmic transportation by disrupting the integrity of nuclear pore complex. One of the major cytoskeletal elements, actin and its dynamics, regulate the dendritic spine structure and functions; impairments have been documented towards learning and memory defects. The second major constituent of these cytoskeletal elements, microtubules, are necessary for the delivery of the cargo, like ion channels and receptors at the synaptic membranes, whereas actin-binding protein, i.e., Cofilin's activation form rod-like structures, is involved in the formation of paired helical filaments (PHFs) observed in AD. Also, the glial cells rely on their cytoskeleton to maintain synaptic functionality. Thus, making cytoskeletal elements and their regulation in synaptic structure and function as an important aspect to be focused for better management and targeting AD pathology. This review advocates exploring phytochemicals and Ayurvedic plant extracts against AD by elucidating their neuroprotective mechanisms involving cytoskeletal modulation and enhancing synaptic plasticity. However, challenges include their limited bioavailability due to the poor solubility and the limited potential to cross the blood-brain barrier (BBB), emphasizing the need for targeted strategies to improve therapeuticefficacy.

Crosstalk between tau and alpha7 nicotinic acetylcholine receptors at the synapse

AbstractBackgroundThe failure of cholinergic transmission and the increase of amyloid‐β peptide (Aβ) and tau protein are key players in Alzheimer’s disease (AD). The interaction between α7 nicotinic acetylcholine receptors (α7‐nAChRs) and Aβ has been previously investigated, but few studies have focused on the crosstalk between α7‐nAChRs and tau. We previously reported that Aβ and tau might act independently or concomitantly to impair synaptic plasticity and memory, converging onto common targets such as amyloid precursor protein (APP). Here we investigated the role of α7‐nAChRs on tau‐mediated impairment of synaptic plasticity and memory, tau neuropathology, and their interplay with APP.MethodWe used WT (C57Bl6/J) and α7 KO at different ages. 3XTg models of AD were used as positive controls. We performed electrophysiology to investigate synaptic transmission and plasticity at the CA3‐CA1 synapses from hippocampal slices, and behavioral studies (Novel Object Recognition and Location, Fear Conditioning) to evaluate memory. We used western blot for the expression of APP, tau (Ser199, Ser396, Thr205) and GSK‐3β (Ser9) phosphorylation. Formation of tau paired helical filaments (PHFs) and neurofibrillary tangles (NFTs) were evaluated by immunohistochemistry and Bielschowsky staining. Immunoreactivity for NeuN was measured to assess neuronal loss.ResultWe found that α7 KO mice presented an AD phenotype characterized by the impairment of Long‐Term Potentiation and Paired Pulse Facilitation, and the reduction of different types of memory starting at 12 months of age. This age‐dependent phenotype was paralleled by an increase of APP expression, hyperphosphorylation of tau, and a decrease of GSK‐3β (Ser9) phosphorylation. α7 KO hippocampi presented a marked tau neuropathology with an increase of PHF‐1 immunoreactivity, the presence of PHFs and NFTs, and neuronal loss.ConclusionIn summary, the lack of α7‐nAChRs was sufficient to trigger an AD‐like phenotype characterized by a marked tau pathology. Our data also suggest that the concomitant increase of APP expression might contribute to tau neurotoxicity.

P-Tau Dephosphorylation Measurement on the Basis of Biomimetic Electrochemical-Polymerized Thin Film Modified Eqcm

Alzheimer’s disease (AD) is characterized by the abnormal protein deposit in the brain, as a most general neurodegenerative disorder in senior. Microtubule-associated tau protein was regarded as a critical biomarker for AD in recent years due to its post-translational modification, especially hyper-phosphorylated tau protein, which would render the protein aggregation and result in neuron degeneration and paired helical filaments (PHF) formation [1]. Unbalanced activities between kinase and phosphatase are the main reason of tau protein hyper-phosphorylation, including overexpression of kinase. Research has recorded that kinases inhibitor would reverse the hyper-phosphorylation induced pathology, showing the possible therapeutic strategy with adjusting the amount of phosphorylated sites. However, this indirect method is insufficient to reverse the neurofibrillary tangle and PHF aggregation [2]. A highly effective method of reversing hyper-phosphorylation need to be investigated. Furthermore, to control the phosphorylation of tau protein, a material with good interaction with biomolecules is required.In this study, polyaniline (PANI), as one of organic conducting polymers [3], was selected due to its fascinating properties and unparalleled multifunction, including good interaction with proteins, controllable electrical conductivity, ideal stability during electrochemical reactions, and changeable nanostructures, could meet above demands and offer further more bio-applications [4]. Through the electrochemical reaction-driven exchange of ions during oxidation and reduction, PANI mimics change in functional biological organs [5] and plays a key role to create a state-of-the-art biomimetic hippocampus.A new in vitro experimental architecture was developed based on electrical quartz crystal microbalance (EQCM). First, tau protein was adsorbed on PANI-modified electrodes by electrostatic interaction to mimic captured process on the hippocampus. Then, both the constant-potential method (current-time curve) and Cyclic Voltammetry (CV) were introduced to investigate the electrochemically-driven dephosphorylation-related process and mechanism following the found optimal potential and stimulation time.As the result, the feasibility of electrochemically-driven dephosphorylation was confirmed. By establishing the relationship between resonant frequency and mass change, this hippocampus-inspired biomimetic polymeric EQCM electrode could detect a trace amount of phosphate and whole protein adsorbed and desorbed to explore the possibility of a non-invasive therapeutic strategy for AD.[1]Alonso, A. D. C., Grundke-Iqbal, I., & Iqbal, K. "Alzheimer's disease hyperphosphorylated tau sequesters normal tau into tangles of filaments and disassembles microtubules," Nature medicine, 2(7), 783-787. (1996).[2]Engel, T., Goñi‐Oliver, P., Lucas, J. J., Avila, J., & Hernández, F., "Chronic lithium administration to FTDP-17 tau and GSK‐3β overexpressing mice prevents tau hyperphosphorylation and neurofibrillary tangle formation, but pre-formed neurofibrillary tangles do not revert," Journal of neurochemistry, 99(6), 1445-1455. (2006).[3]Higgins, M. J., Molino, P. J., Yue, Z., & Wallace, G. G. (2012). Organic conducting polymer–protein interactions. Chemistry of Materials, 24(5), 828-839.[4]Huang, L., Hu, J., Lang, L., Wang, X., Zhang, P., Jing, X., ... & Wei, Y. (2007). Synthesis and characterization of electroactive and biodegradable ABA block copolymer of polylactide and aniline pentamer. Biomaterials, 28(10), 1741-1751.[5] Higgins, M. J., Molino, P. J., Yue, Z., & Wallace, G. G. (2012). Organic conducting polymer–protein interactions. Chemistry of Materials, 24(5), 828-839. Figure 1

Suvorexant Acutely Decreases Tau Phosphorylation and Aβ in the Human CNS.

In Alzheimer's disease, hyperphosphorylated tau is associated with formation of insoluble paired helical filaments that aggregate as neurofibrillary tau tangles and are associated with neuronal loss and cognitive symptoms. Dual orexin receptor antagonists decrease soluble amyloid-β levels and amyloid plaques in mouse models overexpressing amyloid-β, but have not been reported to affect tau phosphorylation. In this randomized controlled trial, we tested the acute effect of suvorexant, a dual orexin receptor antagonist, on amyloid-β, tau, and phospho-tau. Thirty-eight cognitively unimpaired participants aged 45 to 65 years were randomized to placebo (N=13), suvorexant 10 mg (N=13), and suvorexant 20 mg (N=12). Six milliliters of cerebrospinal fluid were collected via an indwelling lumbar catheter every 2 hours for 36 hours starting at 20:00. Participants received placebo or suvorexant at 21:00. All samples were processed and measured for multiple forms of amyloid-β, tau, and phospho-tau via immunoprecipitation and liquid chromatography-mass spectrometry. The ratio of phosphorylated-tau-threonine-181 to unphosphorylated-tau-threonine-181, a measure of phosphorylation at this tau phosphosite, decreased ~10% to 15% in participants treated with suvorexant 20 mg compared to placebo. However, phosphorylation at tau-serine-202 and tau-threonine-217 were not decreased by suvorexant. Suvorexant decreased amyloid-β ~10% to 20% compared to placebo starting 5 hours after drug administration. In this study, suvorexant acutely decreased tau phosphorylation and amyloid-β concentrations in the central nervous system. Suvorexant is approved by the US Food and Drug Administration to treatment insomnia and may have potential as a repurposed drug for the prevention of Alzheimer's disease, however, future studies with chronic treatment are needed. ANN NEUROL 2023.

Structural Analysis and Conformational Dynamics of Short Helical Hyperphosphorylated Segments of Tau Protein (Sequence 254-290) in Alzheimer's Disease: A Molecular Dynamics Simulation Study.

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder whose early diagnosis leads to a chance for successful treatment and decreases the side effects. Hyperphosphorylation of tau proteins is a pathological hallmark of AD that causes it to lose its attachment ability to the microtubules. Alteration of tau structure due to its hyperphosphorylation is an exciting challenge regarding AD treatments. Here, we aimed to examine the structural alterations of short helical segments of tau protein with one to three phosphorylated sites by molecular dynamics simulation. Results indicated that the interaction of two similar segments with three phosphorylated sites (P-Ser262, 285, and 289) formed a compact and more stable structure than the one phosphorylated site complex (P-Ser262). Moreover, due to the high dynamics of the P-Ser262 complex, several structures were made with different conformational dynamics, but there was only one stable cluster of the P-Ser262, 285, and 289 complex during simulation. It seems that the P-Ser262, 285, and 289 complex plays an important role in the formation of paired helical filaments (PHFs) by forming a stable dimer. Generally, it is important to identify how structural features of segments in tau protein change when the phosphorylated sites increase from one to three sites and their effects on the formation of PHFs for drug design and diagnostic biomarkers.

Protective effects of a small molecule inhibitor ligand against hyperphosphorylated tau-induced mitochondrial and synaptic toxicities in Alzheimer disease

The purpose of our study is to understand the protective effects of small molecule ligands for phosphorylated tau (p-tau) in Alzheimer's disease (AD) progression. Many reports show evidence that phosphorylated tau is reported to be an important contributor to the formation of paired helical filaments (PHFs) and neurofibrillary tangles (NFTs) in AD neurons. In AD, glycogen synthase kinase-3 beta (GSK3β), cyclin-dependent kinase-5 and dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A), are the three important kinases responsible for tau hyperphosphorylation. Currently, there are no drugs and/or small molecules that reduce the toxicity of phosphorylated tau in AD. In the present study, we rationally selected and validated small molecule ligands that bind to the phosphorylated tau at SER23 (Ser 285) and TYR44 (Tyr310). We also assessed the molecular dynamics and validated molecular docking sites for the three best ligands. Based on the best docking scores -8.09, -7.9 and -7.8kcal/mol, we found that ligand 1 binds to key hyperphosphorylation residues of phosphorylated tau that inhibit abnormal PHF-tau, DYRK1A and GKS3β that reduce phosphorylated tau levels in AD. Using biochemical, molecular, immunoblotting, immunofluorescence and transmission electron microscopy analyses, we studied the ligand 1 inhibition as well as mitochondrial and synaptic protective effects in immortalized primary hippocampal neuronal (HT22) cells. We found interactions between NAT10-262501 (ligand 1) and phosphorylated tau at key phosphorylation sites and these ligand-based inhibitions decreased PHF-tau, DYRK1A and GSK3β levels. We also found increased mitochondrial biogenesis, mitochondrial fusion and synaptic activities and reduced mitochondrial fission in ligand 1-treated mutant tau HT22 cells. Based on these results, we cautiously conclude that phosphorylated tau NAT10-262501 (ligand 1) reduces hyperphosphorylation of tau based GKS3β and CDK5 kinase regulation in AD, and aids in the maintenance of neuronal structure, mitochondrial dynamics and biogenesis with a possible therapeutic drug target for AD.

Tau Protein Interaction Partners and Their Roles in Alzheimer's Disease and Other Tauopathies.

Tau protein plays a critical role in the assembly, stabilization, and modulation of microtubules, which are important for the normal function of neurons and the brain. In diseased conditions, several pathological modifications of tau protein manifest. These changes lead to tau protein aggregation and the formation of paired helical filaments (PHF) and neurofibrillary tangles (NFT), which are common hallmarks of Alzheimer’s disease and other tauopathies. The accumulation of PHFs and NFTs results in impairment of physiological functions, apoptosis, and neuronal loss, which is reflected as cognitive impairment, and in the late stages of the disease, leads to death. The causes of this pathological transformation of tau protein haven’t been fully understood yet. In both physiological and pathological conditions, tau interacts with several proteins which maintain their proper function or can participate in their pathological modifications. Interaction partners of tau protein and associated molecular pathways can either initiate and drive the tau pathology or can act neuroprotective, by reducing pathological tau proteins or inflammation. In this review, we focus on the tau as a multifunctional protein and its known interacting partners active in regulations of different processes and the roles of these proteins in Alzheimer’s disease and tauopathies.

Epigallocatechin-3-gallate modulates Tau Post-translational modifications and cytoskeletal network.

Background: Alzheimer’s disease is a type of dementia denoted by progressive neuronal death due to the accumulation of proteinaceous aggregates of Tau. Post-translational modifications like hyperphosphorylation, truncation, glycation, etc. play a pivotal role in Tau pathogenesis. Glycation of Tau aids in paired helical filament formation and abates its microtubule-binding function. The chemical modulators of Tau PTMs, such as kinase inhibitors and antibody-based therapeutics, have been developed, but natural compounds, as modulators of Tau PTMs are not much explored.Materials and Methods: We applied biophysical and biochemical techniques like fluorescence kinetics, oligomerization analysis and transmission electron microscopy to investigate the impact of EGCG on Tau glycation in vitro. The effect of glycation on cytoskeleton instability and its EGCG-mediated rescue were studied by immunofluorescence microscopy in neuroblastoma cells.Results: EGCG inhibited methyl glyoxal (MG)-induced Tau glycation in vitro. EGCG potently inhibited MG-induced advanced glycation endproducts formation in neuroblastoma cells as well modulated the localization of AT100 phosphorylated Tau in the cells. In addition to preventing the glycation, EGCG enhanced actin-rich neuritic extensions and rescued actin and tubulin cytoskeleton severely disrupted by MG. EGCG maintained the integrity of the Microtubule Organizing Center (MTOC) stabilized microtubules by Microtubule-associated protein RP/EB family member 1 (EB1).Conclusions: We report EGCG, a green tea polyphenol, as a modulator of in vitro methylglyoxal-induced Tau glycation and its impact on reducing advanced glycation end products in neuroblastoma cells. We unravel unprecedented function of EGCG in remodeling neuronal cytoskeletal integrity.

Biophysical Characterization of Covalently Modified Protein Tau: Oligomers, Aggregation, and Tubulin Interactions

The molecular interactions that drive the onset and spread of Alzheimer's disease (AD) remain poorly understood. In AD, tau neurofibrillary tangles correlate with cognitive decline and have historically been associated with tau's toxicity. Recently, tau oligomers have been implicated as stable toxic subunits that contribute to the formation of paired helical filaments and eventual neurofibrillary tangles. Covalently modified tau is a major component of neurofibrillary tangles and plays a significant role in tau's ability to dissociate from microtubules, aggregate, and become toxic. Elucidating the effects of covalent modifications on oligomer formation could aide our understanding of the molecular mechanisms behind tau's toxicity. The complexity of tau, partially due to its intrinsically disordered nature, allows for phosphorylation, acetylation, and succinylation to occur at multiple sites and influence the physiological and pathological roles of tau. Specific residue modifications can play a major role in modulating tau function and aggregation. This has led to the use of mimics in cell, mouse, and in-vitro models of tau pathology. Site-specific mimics of phosphorylation, acetylation, and succinylation can aide our ability to biophysically characterize the effect of covalently modified tau on oligomer formation, aggregation propensity, and tubulin interactions; Ultimately aiding our understanding of tau function and structure. We apply nuclear magnetic resonance, fluorescence, and light scattering to show that covalently modified tau mimics of phosphorylation, acetylation, and succinylation perturb tau-T2R interactions (a model of tau-tubulin interactions), induce filament formation, and form stable toxic oligomers with different levels of efficiency. Our results reinforce phosphorylation as a contributor to filament formation, acetylation as a driver of oligomer formation, and introduces tau succinylation as a novel modifier of tau structure and function.

Zinc Induces Temperature-Dependent Reversible Self-Assembly of Tau

Tau is an intrinsically disordered microtubule-associated protein that is implicated in several neurodegenerative disorders called tauopathies. In these diseases, Tau is found in the form of intracellular inclusions that consist of aggregated paired helical filaments (PHFs) in neurons. Given the importance of this irreversible PHF formation in neurodegenerative disease, Tau aggregation has been extensively studied. Several different factors, such as mutations or post translational modifications, have been shown to influence the formation of late-stage non-reversible Tau aggregates. It was recently shown that zinc ions accelerated heparin-induced oligomerization of Tau constructs. Indeed, in vitro studies of PHFs have usually been performed in the presence of additional co-factors, such as heparin, in order to accelerate their formation. Using turbidimetry, we investigated the impact of zinc ions on Tau in the absence of heparin and found that zinc is able to induce a temperature-dependent reversible oligomerization of Tau. The obtained oligomers were not amyloid-like and dissociated instantly following zinc chelation or a temperature decrease. Finally, a combination of isothermal titration calorimetry and dynamic light scattering experiments showed zinc binding to a high-affinity binding site and three low-affinity sites on Tau, accompanied by a change in Tau folding. Altogether, our findings stress the importance of zinc in Tau oligomerization. This newly identified Zn-induced oligomerization mechanism may be a part of a pathway different of and concurrent to Tau aggregation cascade leading to PHF formation.

In Vitro Assay for Studying the Aggregation of Tau Protein and Drug Screening.

Aggregation of tau protein and formation of paired helical filaments is a hallmark of Alzheimer's disease and other tauopathies. Compared to other proteins associated with neurodegenerative diseases, the reported in vitro aggregation kinetics for tau protein are less consistent presenting a relatively high variability. Here we describe the development of an in vitro aggregation assay that mimics the expected steps associated with tau misfolding and aggregation in vivo. The assay uses the longest tau isoform (huTau441) which contains both N-terminal acidic inserts as well as four microtubule binding domains (MBD). The in vitro aggregation is triggered by addition of heparin and followed continuously by thioflavin T fluorescence in a 96 well microplate format. The tau aggregation assay is highly reproducible between different wells, experimental runs and batches of the protein. The aggregation leads to tau PHF-like morphology which is very efficient in seeding the formation of de novo fibrillar structures. In addition to its application in studying the mechanism of tau misfolding and aggregation, the current assay is a robust tool for screening drugs that could interfere with the pathogenesis of tau.

Bilateral Implantation of Shear Stress Modifier in ApoE Knockout Mouse Induces Cognitive Impairment and Tau Abnormalities.

Vascular cognitive impairment (VCI) encompasses all causes of cerebrovascular disease that lead to cognitive decline, or overt dementia, atherosclerotic disease being the most common contributor. However, few rodent models that mimic the pathology of VCI replicated the clinical cerebrovascular atherosclerosis. Here we aimed to investigate the mechanism underlying VCI in an Apolipoprotein E knockout (ApoE-KO) mouse model fed with western style food with implantation of bilateral shear stress modifiers. We established a cognitive decline in spatial learning and memory developed in the bilateral modifier treated mice. Brain imaging and pathological examinations demonstrated reduced glucose intake and neuronal loss in hippocampus. Although no amyloid plaques or neurofibrillary tangles (NFTs) were observed, tau pathology including hyperphosphorylation, paired helical filament formation and pathologic truncation were found at considerable higher extent in the bilateral modifier group 8 weeks post the procedure. In addition, gliosis and microglia activation were confirmed in corpus callosum (CC) and ventral striatum. Thus, this ApoE-KO mouse model faithfully replicates the stenosis of common carotid artery (CCA) and cognitive impairment following atherosclerotic deposition and global cerebral hypoperfusion. The close correlation of cognitive decline and tau pathology indicates the toxic tau species could be at least partially responsible for the neurodegenerative changes induced by the chronic hypoxia/ischemia.

Novel cell-penetrating-amyloid peptide conjugates preferentially kill cancer cells.

The goal of this study was to develop a peptide which could use the toxic effects of amyloid, a substance which is the hallmark of over 25 known human diseases, to selectively kill cancer cells. Here we demonstrate that two separate amyloid-forming hexapeptides, one from the microtubule associated protein Tau involved in formation of paired helical filaments of Alzheimer's disease, and the other an amyloid forming sequence from apolipoprotein A1, when conjugated to a cell penetrating peptide (CPP) sequence, form toxic oligomers which are stable for up to 14 h and able to enter cells by a combination of endocytosis and transduction. The amyloid peptide conjugates showed selective cytotoxicity to breast cancer, neuroblastoma and cervical cancer cells in culture compared to normal cells. Fluorescence imaging experiments showed the CPP-amyloid peptide oligomers formed intracellular fibrous amyloid, visible in the endosomes/lysosomes, cytosol and nucleus with thioflavin S (ThS) staining. Further experiments with rhodamine-conjugated Dextran, propidium iodide (PI), and acridine orange (AO) suggested the mechanism of cell death was the permeability of the lysosomal membrane brought about by the formation of amyloid pores. Cytotoxicity could be abrogated by inhibitors of lysosomal hydrolases, consistent with a model where lysosomal hydrolases leak into the cytosol and induce cytotoxicity in subsequent downstream steps. Taken together, our data suggest that CPP-amyloid peptide conjugates show potential as a new class of anti-cancer peptides (ACPs).

Resveratrol induces dephosphorylation of Tau by interfering with the MID1-PP2A complex

The formation of paired helical filaments (PHF), which are composed of hyperphosphorylated Tau protein dissociating from microtubules, is one of the pathological hallmarks of Alzheimer’s disease (AD) and other tauopathies. The most important phosphatase that is capable of dephosphorylating Tau at AD specific phospho-sites is protein phosphatase 2 A (PP2A). Here we show that resveratrol, a polyphenol, significantly induces PP2A activity and reduces Tau phosphorylation at PP2A-dependent epitopes. The increase in PP2A activity is caused by decreased expression of the MID1 ubiquitin ligase that mediates ubiquitin-specific modification and degradation of the catalytic subunit of PP2A when bound to microtubules. Interestingly, we further show that MID1 expression is elevated in AD tissue. Our data suggest a key role of MID1 in the pathology of AD and related tauopathies. Together with previous studies showing that resveratrol reduces β-amyloid toxicity they also give evidence of a promising role for resveratrol in the prophylaxis and therapy of AD.

Pittsburgh Compound-B (PiB) binds amyloid β-protein protofibrils.

The neuropathology of Alzheimer's disease (AD) includes amyloid plaque formation by the amyloid β-protein (Aβ) and intracellular paired helical filament formation by tau protein. These neuropathogenetic features correlate with disease progression and have been revealed in brains of AD patients using positron emission tomography (PET). One of the most useful positron emission tomography imaging agents has been Pittsburgh Compound-B (PiB). However, since its introduction in 2002, substantial evidence has accumulated suggesting that Aβ oligomerization and protofibril formation, rather than fibril formation per se, may be the more important pathogenetic event in AD. Detecting protofibrils and oligomeric forms of Aβ thus may be of value. We report here the results of experiments to determine whether PiB binds to oligomers or protofibrils formed by Aβ40 and Aβ42. We observed strong binding to Aβ42 fibrils, significant binding to protofibrils, and weaker binding to Aβ42 oligomers. PiB also binds Aβ40 fibrils, but its binding to Aβ40 protofibrils and oligomers is substantially lower than for that observed for Aβ42.

Comprehensive Quantitative Profiling of Tau and Phosphorylated Tau Peptides in Cerebrospinal Fluid by Mass Spectrometry Provides New Biomarker Candidates.

Aberrant tau phosphorylation is a hallmark in Alzheimer's disease (AD), believed to promote formation of paired helical filaments, the main constituent of neurofibrillary tangles in the brain. While cerebrospinal fluid (CSF) levels of total tau and tau phosphorylated at threonine residue 181 (pThr181) are established core biomarkers for AD, the value of alternative phosphorylation sites, which may have more direct relevance to pathology, for early diagnosis is not yet known, largely due to their low levels in CSF and lack of standardized detection methods. To overcome sensitivity limitations for analysis of phosphorylated tau in CSF, we have applied an innovative mass spectrometry (MS) workflow, TMTcalibratortrademark, to enrich and enhance the detection of phosphoproteome components of AD brain tissue in CSF, and enable the quantitation of these analytes. We aimed to identify which tau species present in the AD brain are also detectable in CSF and which, if any, are differentially regulated with disease. Over 75% coverage of full-length (2N4R) tau was detected in the CSF with 47 phosphopeptides covering 31 different phosphorylation sites. Of these, 11 phosphopeptides were upregulated by at least 40%, along with an overall increase in tau levels in the CSF of AD patients relative to controls. Use of the TMTcalibratortrademark workflow dramatically improved our ability to detect tau-derived peptides that are directly related to human AD pathology. Further validation of regulated tau peptides as early biomarkers of AD is warranted and is currently being undertaken.

Phospho-Tau and Cognitive Decline in Alzheimer's Disease. Commentary: Tau in physiology and pathology.

In the brain, abnormal phosphorylation of tau leads to the formation of paired helical filaments, which are the main components of intraneuronal neurofibrillary tangles (NFT) (Grundke-Iqbal et al., 1986) that are characteristic of Alzheimer's disease (AD) and other tauopathies (Lee et al., 2001). There is an intense debate about the relationship of phospho-tau and the typical cognitive deficits in AD (Castellani et al., 2008) but, owing to the limited data that is available about synaptic circuits in the normal human brain and in that of the AD patient, the basic mechanism/s of cognitive deterioration are still a mystery. The article by Wang and Mandelkow (2016) presents a timely review of the roles of tau in physiology and disease. However, the role of hyperphosphorylation of tau in dendritic pathology is not fully addressed. Wang and Mandelkow concluded that NFT are probably unrelated to cognitive impairment, mainly based on studies using several lines of mice transgenic for wild-type or mutant human tau. However, in a previous study assessing the possible alterations to dendritic spines in pyramidal cells from AD patients (Merino-Serrais et al., 2013), we found a remarkable loss of dendritic spines from pyramidal cells containing NFT. Since pyramidal neurons represent the principal building blocks of the cerebral cortex and dendritic spines are the main postsynaptic elements of cortical excitatory synapses and are fundamental structures in memory, learning, and cognition (DeFelipe, 2015), these alterations constitute what we think is an important early event in the pathogenesis of AD. We used intracellular injections of Lucifer yellow (LY) in fixed brain tissue from AD patients to visualize and reconstruct dendritic arbors of pyramidal neurons—using high-resolution tile scan stacks of confocal microscopy images—to compare neurons that were free of signs of any neurofibrillary pathology with those showing either diffuse phospho-tau (putative pre-tangle state) or aggregated tau NFT. Following injection in the parahippocampal cortex and CA1, the sections were immunostained for LY and phospho-tau. In the so-called putative “pre-tangle” stage, the dendritic trees of pyramidal neurons were unchanged (pattern I; Figure 1, Panel 1). In the presence of well-developed NFT, however, dendritic spine loss was obvious (pattern IIb; Figure 1, Panel 2). In cases with an intermediate state of neurofibrillary pathology (pattern IIa), the loss of dendritic spines was more variable. Importantly, we compared neighboring cells with and without neurofibrillary pathology (see A, B in Figure 1, Panel 1) to avoid confounding factors such as: (1) morphological differences in the structure of pyramidal cells due to regional specializations (i.e., pyramidal cells in different cortical regions and layers may show morphological differences); (2) high inter-individual variability (sex, age, medical treatment, etc.,) factors that could affect brain structure; and (3) the highly variable course of AD, as the neuropathological changes are not homogenous among patients or in different regions of the brain of the same patient, giving rise to variation in the alterations to cortical circuits. Figure 1 Panel 1. Neurons and dendrites in the parahippocampal cortex of patient P9 injected with LY whose soma is free of phospho-tau PHF-tauAT8-ir (PHF-tauAT8-; A–D) or contains phospho-tau (PHF-tauAT8+ in the putative pre-tangle state (Pattern I; E–H) ... We concluded that the presence of phospho-tau in neurons does not necessarily mean that they suffer severe and irreversible effects as thought previously, but rather the characteristic cognitive impairment in AD is likely to depend on the relative number of neurons that have well-developed tangles. Certainly, these observations differ from those of studies on transgenic mouse brains, where human mutant tau was overexpressed. However, this discrepancy could be explained by the fact that transgenic mouse brains do not reproduce all features of AD found in humans. Furthermore, there are many molecular and structural differences between mouse and human brain. Thus, extrapolation of data obtained in mice and humans is rather difficult and possible mismatches between the two species should be taken into consideration when dealing with animal models of AD.

Difficulties associated with the structural analysis of proteins susceptible to form aggregates: The case of Tau protein as a biomarker of Alzheimer's disease.

Mass spectrometry coupled with bioaffinity separation techniques is considered a powerful tool for studying protein interactions. This work is focused on epitope analysis of tau protein, which contains two VQIXXK aggregation motifs regarded as crucial elements in the formation of paired helical filaments, the main pathological characteristics of Alzheimer's disease. To identify major immunogenic structures, the epitope extraction technique utilizing protein fragmentation and magnetic microparticles functionalized with specific antibodies was applied. However, the natural adhesiveness of some newly generated peptide fragments devalued the experimental results. Beside presumed peptide fragment specific to applied monoclonal anti-tau antibodies, the epitope extraction repeatedly revealed inter alia tryptic fragment 299-HVPGGGSVQIVYKPVDLSK-317 containing the fibril-forming motif 306-VQIVYK-311. The tryptic fragment pro-aggregation and hydrophobic properties that might contribute to adsorption phenomenon were examined by Thioflavin S and reversed-phase chromatography. Several conventional approaches to reduce the non-specific fragment sorption onto the magnetic particle surface were performed, however with no effect. To avoid methodological complications, we introduced an innovative approach based on altered proteolytic digestion. Simultaneous fragmentation of tau protein by two immobilized proteases differing in the cleavage specificity (TPCK-trypsin and α-chymotrypsin) led to the disruption of motif responsible for undesirable adhesiveness and enabled us to obtain undistorted structural data.

β-Secretase 1's Targeting Reduces Hyperphosphorilated Tau, Implying Autophagy Actors in 3xTg-AD Mice.

β-site APP cleaving enzyme 1 (BACE1) initiates APP cleavage, which has been reported to be an inducer of tau pathology by altering proteasome functions in Alzheimer’s disease (AD). However, the exact relationship between BACE1 and PHF (Paired Helical Filaments) formation is not clear. In this study, we confirm that BACE1 and Hsc70 are upregulated in the brains of AD patients, and we demonstrate that both proteins show enhanced expression in lipid rafts from AD-affected triple transgenic mouse brains. BACE1 targeting increased Hsc70 levels in the membrane and cytoplasm fractions and downregulated Hsp90 and CHIP in the nucleus in the hippocampi of 3xTg-AD mice. However, these observations occurred in a proteasome-independent manner in vitro. The BACE1miR-induced reduction of soluble hyperphosphorylated tau was associated with a decrease in MAPK activity. However, the BACE1 RNAi-mediated reduction of hyperphosphorylated tau was only blocked by 3-MA (3-methyladenine) in vitro, and it resulted in the increase of Hsc70 and LAMP2 in lipid rafts from hippocampi of 3xTg-AD mice, and upregulation of survival and homeostasis signaling. In summary, our findings suggest that BACE1 silencing neuroprotects reducing soluble hyperphosphorylated tau, modulating certain autophagy-related proteins in aged 3xTg-AD mice.