Insoluble Paired Helical Filaments Research Articles

Increased retention of tau PET ligand [18F]-AV1451 in Alzheimer\u2019s Disease Psychosis

Psychosis in Alzheimer’s disease (AD) represents a distinct disease subtype with a more rapid progression of illness evidenced by an increased velocity of cognitive decline and a hastened mortality. Previous biomarker and post-mortem studies have implicated tau neuropathology as a possible mediator of the accelerated decline in AD psychosis. Tau positron emission tomography (PET) neuroimaging provides the opportunity to evaluate tau pathology in-vivo, so that clinical symptomatology can be correlated with disease pathology. [18F]-AV1451 (Flortaucipir) is a PET ligand with high affinity for insoluble paired-helical filaments (PHFs) of hyperphosphorylated tau. In order to determine whether the development of psychosis and worsened prognosis in AD is associated with an increased burden of tau pathology that can be identified with tau imaging, we identified subjects within the Alzheimer’s disease neuroimaging initiative (ADNI) who had [18F]-AV1451 imaging at baseline and became psychotic over the course of the study (N = 17) and matched them 1:3 for gender, age, and education to subjects who had [18F]-AV1451 imaging at baseline and did not become psychotic (N = 50). We compared baseline [18F]-AV1451 retention, in addition to cognitive and functional baseline and longitudinal change, in those who became psychotic over the course of participation in ADNI with those who did not. Results suggest that increases in tau pathology in frontal, medial temporal, and occipital cortices, visualized with [18F]-AV1451 binding, are associated with psychosis and a more rapid cognitive and functional decline.

Molecular Processing of Tau Protein in Progressive Supranuclear Palsy: Neuronal and Glial Degeneration.

Background:Alzheimer’s disease (AD) and progressive supranuclear palsy (PSP) are examples of neurodegenerative diseases, characterized by abnormal tau inclusions, that are called tauopathies. AD is characterized by highly insoluble paired helical filaments (PHFs) composed of tau with abnormal post-translational modifications. PSP is a neurodegenerative disease with pathological and clinical heterogeneity. There are six tau isoforms expressed in the adult human brain, with repeated microtubule-binding domains of three (3R) or four (4R) repeats. In AD, the 4R:3R ratio is 1:1. In PSP, the 4R isoform predominates. The lesions in PSP brains contain phosphorylated tau aggregates in both neurons and glial cells.Objective:Our objective was to evaluate and compare the processing of pathological tau in PSP and AD.Methods:Double and triple immunofluorescent labeling with antibodies to specific post-translational tau modifications (phosphorylation, truncation, and conformational changes) and thiazin red (TR) staining were carried out and analyzed by confocal microscopy.Results:Our results showed that PSP was characterized by phosphorylated tau in neurofibrillary tangles (NFTs) and glial cells. Tau truncated at either Glu391 or Asp421 was not observed. Extracellular NFTs (eNFTs) and glial cells in PSP exhibited a strong affinity for TR in the absence of intact or phosphorylated tau.Conclusion:Phosphorylated tau was as abundant in PSP as in AD. The development of eNFTs from both glial cells and neuronal bodies suggests that truncated tau species, different from those observed in AD, could be present in PSP. Additional studies on truncated tau within PSP lesions could improve our understanding of the pathological processing of tau and help identify a discriminatory biomarker for AD and PSP.

Degradation and Transmission of Tau by Autophagic-Endolysosomal Networks and Potential Therapeutic Targets for Tauopathy.

Tauopathies are a class of neurodegenerative diseases, including Alzheimer’s disease (AD), Frontotemporal Dementia (FTD), Progressive Supranuclear Palsy (PSP), Corticobasal Degeneration (CBD), and many others where microtubule-associated protein tau (MAPT or tau) is hyperphosphorylated and aggregated to form insoluble paired helical filaments (PHFs) and ultimately neurofibrillary tangles (NFTs). Autophagic-endolysosomal networks (AELN) play important roles in tau clearance. Excessive soluble neurotoxic forms of tau and tau hyperphosphorylated at specific sites are cleared through the ubiquitin-proteasome system (UPS), Chaperon-mediated Autophagy (CMA), and endosomal microautophagy (e-MI). On the other hand, intra-neuronal insoluble tau aggregates are often degraded within lysosomes by macroautophagy. AELN defects have been observed in AD, FTD, CBD, and PSP, and lysosomal dysfunction was shown to promote the cleavage and neurotoxicity of tau. Moreover, several AD risk genes (e.g., PICALM, GRN, and BIN1) have been associated with dysregulation of AELN in the late-onset sporadic AD. Conversely, tau dissociation from microtubules interferes with retrograde transport of autophagosomes to lysosomes, and that tau fragments can also lead to lysosomal dysfunction. Recent studies suggest that tau is not merely an intra-neuronal protein, but it can be released to brain parenchyma via extracellular vesicles, like exosomes and ectosomes, and thus spread between neurons. Extracellular tau can also be taken up by microglial cells and astrocytes, either being degraded through AELN or propagated via exosomes. This article reviews the complex roles of AELN in the degradation and transmission of tau, potential diagnostic/therapeutic targets and strategies based on AELN-mediated tau clearance and propagation, and the current state of drug development targeting AELN and tau against tauopathies.

Deletion of endogenous Tau proteins is not detrimental in Drosophila.

Human Tau (hTau) is a highly soluble and natively unfolded protein that binds to microtubules within neurons. Its dysfunction and aggregation into insoluble paired helical filaments is involved in the pathogenesis of Alzheimer’s disease (AD), constituting, together with accumulated β-amyloid (Aβ) peptides, a hallmark of the disease. Deciphering both the loss-of-function and toxic gain-of-function of hTau proteins is crucial to further understand the mechanisms leading to neurodegeneration in AD. As the fruit fly Drosophila melanogaster expresses Tau proteins (dTau) that are homologous to hTau, we aimed to better comprehend dTau functions by generating a specific tau knock-out (KO) fly line using homologous recombination. We observed that the specific removal of endogenous dTau proteins did not lead to overt, macroscopic phenotypes in flies. Indeed, survival, climbing ability and neuronal function were unchanged in tau KO flies. In addition, we did not find any overt positive or negative effect of dTau removal on human Aβ-induced toxicity. Altogether, our results indicate that the absence of dTau proteins has no major functional impact on flies, and suggests that our tau KO strain is a relevant model to further investigate the role of dTau proteins in vivo, thereby giving additional insights into hTau functions.

Protective Effect of Tat PTD-Hsp27 Fusion Protein on Tau Hyperphosphorylation Induced by Okadaic Acid in the Human Neuroblastoma Cell Line SH-SY5Y.

Alzheimer’s disease (AD) is an age-related disorder that causes a loss of brain function. Hyperphosphorylation of tau and the subsequent formation of intracellular neurofibrillary tangles (NFTs) are implicated in the pathogenesis of AD. Hyperphosphorylated tau accumulates into insoluble paired helical filaments that aggregate into NFTs; therefore, regulation of tau phosphorylation represents an important treatment approach for AD. Heat shock protein 27 (Hsp27) plays a specific role in human neurodegenerative diseases; however, few studies have examined its therapeutic effect. In this study, we induced tau hyperphosphorylation using okadaic acid, which is a protein phosphatase inhibitor, and generated a fusion protein of Hsp27 and the protein transduction domain of the HIV Tat protein (Tat-Hsp27) to enhance the delivery of Hsp27. We treated Tat-Hsp27 to SH-SY5Y neuroblastoma cells for 2 h; the transduction level was proportional to the Tat-hsp27 concentration. Additionally, Tat-Hsp27 reduced the level of hyperphosphorylated tau and protected cells from apoptotic cell death caused by abnormal tau aggregates. These results reveal that Hsp27 represents a valuable protein therapeutic for AD.Electronic supplementary materialThe online version of this article (doi:10.1007/s10571-015-0199-1) contains supplementary material, which is available to authorized users.

Hsp27 Reduces Phosphorylated Tau and Prevents Cell Death in the Human Neuroblastoma Cell Line SH-SY5Y

The two major symptoms characterizing Alzheimer's disease are the formation of amyloid- extracellular deposits in the form of senile plaques and intracellular neurofibrillary tangles (NFTs) that consist of pathological hyperphosphorylated tau protein aggregated into insoluble paired helical filaments (PHFs). Neurons of the central nervous system have appreciable amounts of tau protein, a microtubule-associated protein. To maintain an optimal operation of nerves, the microtubules are stabilized, which is necessary to support cell structure and cellular processes. When the modified tau protein becomes dysfunctional, the cells containing misfolded tau cannot maintain cell structure. One of the pathological hallmarks of Alzheimer's disease is hyperphosphorylated tau protein. This paper shows that the small heat shock protein from humans (Hsp27) reduces hyperphosphorylated tau and prevents hyperphosphorylated tau-induced cell death of the human neuroblastoma cell line SH-SY5Y.

Proteasome–caspase–cathepsin sequence leading to tau pathology induced by prostaglandin J2 in neuronal cells

Neurofibrillary tangles (NFT) are a hallmark of Alzheimer's disease. The major neurofibrillary tangle component is tau that is truncated at Asp421 (Deltatau), hyperphosphorylated and aggregates into insoluble paired helical filaments. Alzheimer's disease brains also exhibit signs of inflammation manifested by activated astrocytes and microglia, which produce cytotoxic agents among them prostaglandins. We show that prostaglandin (PG) J2, an endogenous product of inflammation, induces caspase-mediated cleavage of tau, generating Deltatau, an aggregation prone form known to seed tau aggregation prior to neurofibrillary tangle formation. The initial event observed upon PGJ2-treatment of human neuroblastoma SK-N-SH cells was the build-up of ubiquitinated (Ub) proteins indicating an early disruption of the ubiquitin-proteasome pathway. Apoptosis kicked in later, manifested by caspase activation and caspase-mediated cleavage of tau at Asp421 and poly (ADP-ribose) polymerase. Furthermore, cathepsin inhibition stabilized Deltatau suggesting its lysosomal clearance. Upon PGJ2-treatment tau accumulated in a large perinuclear aggregate. In rat E18 cortical neuronal cultures PGJ2-treatment also generated Deltatau detected in dystrophic neurites. Levels of Deltatau were diminished by caspase 3 knockdown using siRNA. PGD2, the precursor of PGJ2, produced some Deltatau. PGE2 generated none. Our data suggest a potential sequence of events triggered by the neurotoxic product of inflammation PGJ2 leading to tau pathology. The accumulation of Ub proteins is an early response. If cells fail to overcome the toxic effects induced by PGJ2, including accumulation of Ub proteins, apoptosis kicks in triggering caspase activation and tau cleavage, the clearance of which by cathepsins could be compromised culminating in tau pathology. Our studies are the first to provide a mechanistic link between inflammation and tau pathology.

Pseudohyperphosphorylation Causing AD-like Changes in Tau Has Significant Effects on Its Polymerization

The microtubule-associated protein tau, in a hyperphosphorylated form, aggregates into insoluble paired-helical filaments (PHFs) in Alzheimer's disease (AD) and other tauopathies. In AD, there is approximately 8 mol of phosphate per mole of tau distributed among approximately 30 PHF phosphorylation sites as compared to 2-3 mol of phosphate per mole in normal brain. In AD, kinases such as glycogen synthase kinase-3beta (GSK-3beta) are believed to be involved in the generation of hyperphosphorylated tau. However, the functional consequences of hyperphosphorylation on the microtubule binding and polymerization of tau are not well understood. To address this question, we have generated pseudohyperphosphorylation mutants consisting of six and seven sites in the proline-rich region and carboxy terminus of tau by amino acid substitution. In addition, several single, double, and triple pseudophosphorylation mutants were also generated. Pseudophosphorylation of tau decreases its affinity for microtubules, and pseudohyperphosphorylated forms of tau do not have significantly decreased levels of microtubule binding as compared to single and double sites. Three pseudohyperphosphorylated forms of tau with altered sodium dodecyl sulfate-polyacrylamide gel electrophoresis migration have a greater effect on its inducer-mediated polymerization, slowing the rate of nucleation and elongation. On the basis of the observations that pseudohyperphosphorylated tau has decreased affinity for microtubules and reduced inducer-initiated rates of nucleation and polymerization, we propose that this combination could be the cause of the increased cytotoxicity of hyperphosphorylated tau in Alzheimer's disease and also explain the potentially beneficial role of tau polymerization and NFT formation.

Comparative Vibrational Spectroscopy of Intracellular Tau and Extracellular Collagen I Reveals Parallels of Gelation and Fibrillar Structure

The N-terminal tau 2-19 peptide undergoes gelation, syneresis, and aggregation over a period of years. These changes may be approximated on a shorter time scale by agitation and partial dehydration. The anomalously enhanced (229 nm) ultraviolet resonance Raman (UVRR) imide II band reveals a common structural feature for gels of nondehydrated tau 2-19 and collagen I and insoluble paired helical filaments (PHFs) and collagen I of weak hydrogen bonding at proline carbonyls. Anomalous UVRR enhancement of amide bands at 229 nm results from gel structure, as demonstrated by increased amide absorption at the red edge for tau 2-19 gel and implies the involvement of water in gel structure. In aged, dehydrated tau 2-19 gel, proline carbonyls lose their bonds to water and tyrosine becomes deprotonated, as demonstrated by UVRR spectroscopy. The Fourier transform infrared (FTIR) amide I band shows that antiparallel beta-sheet structure increases with syneresis in the tau 2-19 hydrogel. The comparison of FTIR results for PHFs with collagen I gel and polyproline demonstrates that the secondary structure of PHFs is polyproline II. One implication of this assignment is that the fibrillation of hydrophilic tau is thermodynamically driven by the entropy gained as hydrogen-bonded water is freed, as for collagen I. The FTIR results also show that peptide domains culled from a longer protein do not necessarily fold into identical secondary structures. A pathological, sequential mechanism of gelation, syneresis, and fibrillation for tau in AD is suggested and is supported by the observation of amorphous neurofibrillary tangle development and fibrillation in vivo.

Identification of microtubule-associated protein tau isoforms in Alzheimer's paired helical filaments.

AD66 proteins derived from sodium dodecylsulfate (SDS) insoluble paired helical filaments (PHF) were isolated from Alzheimer's brain using a purification procedure developed previously in this laboratory, and characterized by immunologic and chemical cleavage methods. AD66 proteins were immunoreactive with antibodies that recognize the amino terminal, tubulin-binding, and carboxy terminal domains of microtubule-associated protein tau indicating the presence of the entire tau sequence in AD66 proteins. These proteins were reactive with antibody 423 that binds to PHF but not human adult tau. Immunologic and chemical cleavage studies indicated that only two of the six tau isoforms were present in these proteins. AD66 proteins were comprised of tau proteins containing only three tubulin binding domains with either a 29 amino acid insert or no amino terminal insert. For comparative purposes, SDS soluble PHF-tau (A68 proteins) was purified from Alzheimer's brains and normal adult tau purified from control brains. Antibody Alz-50 was immunoreactive with PHF-tau or normal tau regardless of alkaline phosphatase treatment while immunoreactivity was only observed with dephosphorylated AD66 proteins. A second phosphorylated epitope on AD66 proteins but not PHF-tau or normal tau proteins was demonstrated with antibody PHF9. These data suggest that AD66 proteins represent a more phosphorylated form of tau than PHF-tau or normal tau proteins. Two-dimensional gel electrophoresis demonstrated that AD66 proteins have higher apparent molecular weights and lower pI values than normal tau, differences possibly due to the greater phosphorylation observed in these proteins.

Quantitative solubilization and analysis of insoluble paired helical filaments from alzheimer disease

In this study, we evaluate the ability of several solvents to solubilize insoluble paired helical filaments (PHF) of Alzheimer disease. Specifically, we use protein extraction and reduction in the volume of insoluble material as quantitative assays to establish solvents of PHF. Using sequential categories of protein solvent to analyze insoluble PHF, only alkali or exhaustive proteolysis are effective in completely solubilizing PHF, while a variety of denaturants are ineffective. Alkali does not affect the phosphorylation state of PHF and complete dephosphorylation of PHF with hydrofluoric acid does not affect PHF solubility. These findings suggest that the ‘hyperphos-sphorylation’ of PHF proteins is not responsible for PHF insolubility. However the in vitro glycation of τ generates PHF that are insoluble in SDS and soluble in alkali. These findings suggest that protein crosslinks, including advanced glycation endproduct-derived crosslinks which were recently described in Alzheimer disease, play a major role in effecting PHF insolubility in vivo.

X-ray probe microanalysis of Alzheimer disease soluble and insoluble paired helical filaments

The paired helical filaments of Alzheimer disease, which have been shown to consist of both soluble and insoluble forms, were examined by X-ray probe microanalysis in order to determine if there existed differences in their elemental composition. The soluble paired helical filaments contained both sulfur and phosphorus, supporting their composition being enriched in a phosphorylated protein. The insoluble paired helical filament core structures, which retained their morphology after extensive protease digestion, contained only a small amount of sulfur over background, which suggests that they are not composed entirely of protein. This significant difference in sulfur and phosphorus indicates a difference in composition between the soluble and insoluble paired helical filaments, and that the paired helical filament core structures may attribute their insolubility to their being predominately non-proteinaceous.

Association of heparan sulfate proteoglycan with the neurofibrillary tangles of Alzheimer's disease

The major intracytoplasmic lesion of Alzheimer's disease is the neurofibrillary tangle (NFT), which is primarily composed of paired helical filaments (PHFs). The mechanism responsible for the formation of PHFs, as well as their insolubility and apparent heterogeneity, is unknown. We found that basic fibroblast growth factor (bFGF) binds to heparinase-sensitive sites in NFTs. bFGF binding is due to a heparan sulfate proteoglycan (HSPG) immunocytochemically identified in NFTs. In the presence of polycations (e.g., Ca2+), HSPG will bind to free carboxyl groups in NFT proteins. HSPG binding may play a role in transforming normal soluble proteins into insoluble PHFs.

Purification and Solubilization of Paired Helical Filaments from Alzheimer Brains

The purpose of the present study was to develop a purification and solubilization method, compatible with current amino acid sequencing techniques, for paired helical filaments (PHFs) derived from patients with Alzheimer's disease. We have developed a mild procedure that subjects conventionally isolated PHFs to Tris/borate/sodium dodecyl sulfate/2-mercaptoethanol electrophoresis and results in the separation of the relatively insoluble PHF structures from both copurifying contaminating proteins and solubilized PHF-associated proteins. At the end of 4.5 h of electrophoresis, the purified insoluble fraction had an amino acid composition that was invariant during subsequent electrophoresis. Electron microscopy revealed an intact PHF structure before and after electrophoresis but no evidence of any other structures in the insoluble fraction, a result consistent with the removal of PHF-associated proteins from the filament structure. Isolated insoluble filament structures displayed an enhanced immunoreactivity with antibodies raised against purified PHFs in other laboratories, when compared with the fraction not subjected to electrophoresis in enzyme-linked immunosorbent assays. Solubilization of the relatively insoluble PHFs was accomplished by extending the time of electrophoresis beyond the 4.5 h required for purification. Additional electrophoresis for 34.5 h solubilized 88% of the purified, relatively insoluble PHFs. This resulted in the identification of four major protein bands between Mr values of approximately 50,000 and 70,000 on sodium dodecyl sulfate-polyacrylamide electrophoresis gel analysis, with a predominant band with an Mr of approximately 66,000. A slow fragmentation of the PHF ultrastructure occurred during this time, as judged by electron microscopy. This purification technique will permit the isolation of consistently reproducible protein fragments from solubilized PHFs, which may be used for subsequent sequence analysis.

Neuronal transformations in Alzheimer's disease.

Brains of nine early and four advanced Alzheimer patients have been investigated, utilizing three approaches to specify the threshold state of Alzheimer's disease (AD). Extensive thin sectioning electron microscopy (EM) of frontal lobe biopsies, correlated with stringent clinical assessment, has demonstrated that the neuronal cytoskeleton undergoes specific transformations into paired helical filament-like (PHF-like) strands, which lead to the formation of the insoluble paracrystalline paired helical filaments (PHFs). The neurofilamentous network (NFN) transformation plays an important role in this process, whereby segregation, posttranslational modifications and reassembly of the modified components through autocrosslinking, and phase transition occur. According to our data, the threshold state can be defined as the state of irreversible segregation and posttranslational modification of the NFN and the microtubule-associated proteins. At this state, therapeutic intervention to reverse the disease process may be possible. The results indicate similarities between the formation of the paracrystals of the PHFs and the formation of the tropomyosin-like crystals of the Hirano bodies. Close relationships among PHFs and smooth endoplasmic reticulum and plasma membrane do exist. Enveloped virus-like particles have been observed in neurons containing PHFs. A possible role of these virus-like particles as an etiological agent for AD is discussed.

Altered structural proteins in plaques and tangles: What do they tell us about the biology of Alzheimer??s disease?

The progressive dysfunction and loss of neurons in Alzheimer's disease (AD) is accompanied by marked structural changes in innumerable neuronal cell bodies and neurites, particularly in limbic and association cortices. Qualitatively indistinguishable neuronal lesions occur in much smaller numbers during normal aging. Highly insoluble paired helical filaments (PHF) and antigenically related straight filaments accumulate in perikaryal tangles and the neurites of neuritic plaques. In addition, PHF antibodies reveal the presence of PHF antigens in many individual cortical neurites not clustered into discrete plaques. Recent studies in several laboratories indicate that altered forms of the microtubule-associated phosphoprotein, tau, are important constituents of PHF. Other neuronal cytoskeletal proteins, particularly microtubule-associated protein 2 and neurofilament, have also been associated with PHF. In contrast, the extracellular amyloid filaments found in the centers of many neuritic plaques and in cortical and meningeal vessels appear to be composed of hydrophobic low molecular weight protein(s) distinct from PHF. A major question for further study regards the cellular origin and role of microvascular amyloid in the degeneration of neurites of multiple neurotransmitter specificities in AD cortex. The widespread neuritic and perikaryal alterations in brain tissue are likely to represent, at least in part, the morphological substrate of cortical dysfunction in AD.

Neuropathological processes in Alzheimer's disease

AbstractDuring the past few years, substantial progress has been made in understanding the neuropathology of Alzheimer's disease (AD) and its late‐life variant, senile dementia of the Alzheimer's type. Neurofibrillary tangles have been shown to be composed of cross‐linked, highly insoluble paired helical filaments. The disease selectively affects certain populations of nerve cells, including those in the amygdala, hippocampus, neocortex, locus coeruleus and basal forebrain cholinergic (Ch) system. Changes in the latter system account, in substantial part, for the decrease in cortical Ch markers which, to date, appear to be the earliest and most consistent abnormalities in the brains of individuals with AD. Neuritic plaques have been shown to be foci of axonal/synaptic pathology consistent with the concept that these changes may be associated with a chronic distal axonopathy involving certain populations of nerve cells. Recent studies in aged nonhuman primates suggest that some of the abnormal axons/terminals in neuritic plaques may be derived from the basal forebrain Ch system. Understanding the biological mechanisms leading to these structural changes is essential if we are to understand the pathogenesis of the clinical manifestations of AD and if we are to design rational therapeutic approaches to treat this disorder.