Amyloid-binding Protein Research Articles

Exploring NSAIDs as anti‐amyloidal agents: A drug repurposing approach to treat Alzheimer’s disease

AbstractBackgroundAlzheimer is a severe, stealthy and progressive neurodegenerative disease. Maximum number of cases are arises after the age of 65 ie. called Late Onset of Alzheimer Disease. AD is characterised by the neuronal cell dysfunctioning / death, shrinkage of brain tissue and progressive cognitive, motor, behavioural impairment. Literature suggest that NSAIDs are able to alter the conformation of Aβ peptides exerting anti‐aggregation activity and induce the expression of amyloid‐binding proteins, e.g., transthyretin, that play an important role in sequestering Aβ peptides and preventing their aggregation.MethodIn‐silico studies are totally based on the computational work, leads to counting receptor and ligand preparation, docking simulations, grid formation and its analysis followed with in‐vitro investigation via Thioflavin T‐amyloid binding assay and protease (Trypsin) digestion assay.ResultThe computational approach provided us naproxen with higher estimated free energy of binding. The drug has the property to traverse the blood brain barrier and hence, it might have the possibility of curing or treating the Alzheimer’s through altering the conformation of Aβ peptides and exerting anti‐aggregation activity.ConclusionNASAIDs may alter the conformation of amyloid beta and exerts anti‐aggregation activity by inducing the expression of amyloid binding proteins.

Undiscovered Roles for Transthyretin: From a Transporter Protein to a New Therapeutic Target for Alzheimer's Disease.

Transthyretin (TTR), an homotetrameric protein mainly synthesized by the liver and the choroid plexus, and secreted into the blood and the cerebrospinal fluid, respectively, has been specially acknowledged for its functions as a transporter protein of thyroxine and retinol (the latter through binding to the retinol-binding protein), in these fluids. Still, this protein has managed to stay in the spotlight as it has been assigned new and varied functions. In this review, we cover knowledge on novel TTR functions and the cellular pathways involved, spanning from neuroprotection to vascular events, while emphasizing its involvement in Alzheimer’s disease (AD). We describe details of TTR as an amyloid binding protein and discuss its interaction with the amyloid Aβ peptides, and the proposed mechanisms underlying TTR neuroprotection in AD. We also present the importance of translating advances in the knowledge of the TTR neuroprotective role into drug discovery strategies focused on TTR as a new target in AD therapeutics.

Structural Switch from Hairpin to Duplex/Antiparallel G-Quadruplex at Single-Nucleotide Polymorphism (SNP) Site of Human Apolipoprotein E (APOE) Gene Coding Region.

A gradual dementia, which leads to the loss of memory and intellectual abilities, is the main characteristics of Alzheimer’s disease. Amyloid-β (Aβ) plaques are the main components that accumulate and form clumps in the brains of people suffering from Alzheimer’s disease. Apolipoprotein E (APOE), an amyloid-binding protein is considered as one of the main genetic risk factor of the late-onset Alzheimer’s disease. Different isoforms of APOE gene named APOE2, APOE3, and APOE4 are known to exist, which differ from each other at certain positions involving single-nucleotide polymorphisms (SNPs). Out of these isoforms, APOE4 increases the risk of developing late-onset Alzheimer’s disease, whereas APOE3 is the most common among the general population. APOE4 differs from the common APOE3 by only one nucleotide at position +2985 (T to C), which results in immense alteration in the structure and function of the APOE gene. A combination of gel electrophoresis (polyacrylamide gel electrophoresis, PAGE), circular dichroism (CD), CD melting, thermal difference spectra and UV-thermal denaturation (TM) techniques was used to investigate the structural polymorphism associated with T → C single-nucleotide polymorphism (SNP) at the GC-rich sequence (d-TGGAGGACGTGTGCGGCCGCCT; APOE22T). Herein, we report that APOE22T DNA sequence switches between hairpin to antiparallel quadruplex from low to high oligomer concentration. On the contrary, its C-counterpart (APOE22C) forms hairpin as well as intermolecular antiparallel duplex structure. This structural change may possibly contribute to the protein recognition pattern, which, in turn, might control the APOE gene expression.

Effect of Bioregulator Isatin on Protein–Protein Interactions Involving Isatin-Binding Proteins

Isatin (2,3-dioxoindol) is an endogenous low-molecular-weight nonpeptide compound with a wide spectrum of biological and pharmacological activities. It is assumed that isatin acts through isatin-binding proteins. To date, more than a hundred of these proteins are known. Having a different structure and cellular and subcellular localization, they belong to different functional groups. Using the surface plasmon resonance technology, we found earlier that isatin affected the profile of intracellular amyloid-binding proteins and changed the stability of protein complexes in the model system. In fact, this indicates the selective effect of isatin on certain protein–protein interactions (PPI) that occur primarily with the participation of isatinbinding proteins. Therefore, we had formulated the hypothesis that isatin could be a regulator of a protein interactome. This study focuses on the verification of this assumption. Size-exclusion chromatography (SEC) profile of the rat liver tissue lysate along with mass-spectrometric protein identification has revealed 20 isatinbinding proteins that participate in the formation of the protein interactome. About 65 and 25% of them are involved in the formation of multimeric protein complexes and homo/heterodimers, respectively, and only 10% are detected as single molecules. The addition of isatin had a multidirectional effect on the profile of about half of the identified isatin-binding proteins. In some cases, the formation of protein complexes was induced, while in other cases the protein complexes were dissociated. This result confirms the hypothesis of the regulatory effect of isatin on certain PPIs. The data of this work in combination with our previous results allowed us to formulate an “interactomics image” of isatin as a bioregulator, which selectively controls both the formation and dissociation of a number of protein complexes. Two new isatin-dependent proteins were found in the work. This indicates that not all potential target proteins of the regulatory effect of isatin had been previously detected. The study of the molecular mechanisms of isatin action on PPI remains a difficult but priority task for future research.

USING EMERGING CEREBROSPINAL FLUID MARKERS TO CHARACTERIZE SUSPECTED NON-ALZHEIMER’S DISEASE PATHOPHYSIOLOGY (SNAP) IN INDIVIDUALS WITH MILD COGNITIVE IMPAIRMENT

The underlying pathophysiology of individuals with suspected non-Alzheimer's disease pathophysiology (SNAP; biomarker profile with neuronal injury without amyloid pathology) is to date still unclear. Our aim was to investigate the role of inflammation and lipid transport in individuals with mild cognitive impairment (MCI) and SNAP using emerging markers in cerebrospinal fluid (CSF). We selected 77 subjects with MCI and 15 cognitively normal (CN) subjects from the European EDAR study. We measured C3a (central to the classical, alternative and lectin complement pathways), the soluble C5b-9 terminal complement complex (TCC), amyloid binding proteins heart-fatty acid binding protein (HFABP), amyloid beta(Aβ)1–42, total tau (t-tau), and phosphorylated tau (p-tau) in CSF, and serum amyloid P (SAP, an acute response protein which activates complement). CN subjects had normal Aβ1–42 and tau. MCI subjects were classified as having low AD likelihood (n=18, normal Aβ1–42 and tau), SNAP (n=14, normal Aβ1–42 and abnormal tau), isolated amyloid pathology (IAP n=18; abnormal Aβ1–42 and normal tau), or high AD likelihood (n=27, abnormal Aβ1–42 and tau). We compared MCI subgroups using ANOVA and correlated emerging and core CSF Alzheimer's disease markers using Spearman (rho). CN individuals had higher levels of C3a than all MCI groups. Individuals with MCI-SNAP and MCI-high AD likelihood had higher levels of HFABP compared to those with MCI-low AD likelihood and MCI-IAP. Individuals with MCI-high AD likelihood had also higher levels of HFABP than CN individuals (see Table 1). In individuals with MCI, C3a correlated with p-tau (r=0.311, p=0.025), and HFABP correlated with Aβ1–42 (r=-0.242, p=0.034), t-tau (r=0.641, p<0.001) and p-tau (r=0.489, p<0.001). Our findings suggest that inflammation plays a similar role in neurodegeneration within all MCI subgroups. Moreover, HFABP levels are related to overall tau-related neurodegeneration in individuals with MCI.

Nucleobindin 1 binds to multiple types of pre-fibrillar amyloid and inhibits fibrillization

During amyloid fibril formation, amyloidogenic polypeptides misfold and self assemble into soluble pre-fibrillar aggregates, i.e., protofibrils, which elongate and mature into insoluble fibrillar aggregates. An emerging class of chaperones, chaperone-like amyloid binding proteins (CLABPs), has been shown to interfere with aggregation of particular misfolded amyloid peptides or proteins. We have discovered that the calcium-binding protein nuclebindin-1 (NUCB1) is a novel CLABP. We show that NUCB1 inhibits aggregation of islet-amyloid polypeptide associated with type 2 diabetes mellitus, a-synuclein associated with Parkinson’s disease, transthyretin V30M mutant associated with familial amyloid polyneuropathy, and Aβ42 associated with Alzheimer’s disease by stabilizing their respective protofibril intermediates. Kinetic studies employing the modeling software AmyloFit show that NUCB1 affects both primary nucleation and secondary nucleation. We hypothesize that NUCB1 binds to the common cross-β-sheet structure of protofibril aggregates to “cap” and stabilize soluble macromolecular complexes. Transmission electron microscopy and atomic force microscopy were employed to characterize the size, shape and volume distribution of multiple sources of NUCB1-capped protofibrils. Interestingly, NUCB1 prevents Aβ42 protofibril toxicity in a cellular assay. NUCB1-stabilized amyloid protofibrils could be used as immunogens to prepare conformation-specific antibodies and as novel tools to develop screens for anti-protofibril diagnostics and therapeutics.

The effect of neuroprotector isatin on binding of some model proteins with beta-amyloid peptide: a biosensor study

The amyloid-beta peptide 1-42 formed during proteolytic processing of the amyloid precursor protein (APP) plays a key role in the development or progression of Alzheimer's disease (AD) and other pathologies associated with formation of protein aggregates in the central nervous system. Recent proteomic profiling of mouse and rat brain preparations by means of beta-amyloid peptide immobilized on Affigel-10 revealed a large group of amyloid-binding proteins (n>80). Many (about 25%) of these proteins were previously identified as isatin-binding proteins. The aim of this study was to validate direct interaction between beta-amyloid peptide and highly purified intact and oxidized peroxiredoxin, M-type pyruvate kinase, alpha-enolase, and the effect of isatin on this interaction. The study performed using SPR-based Biacore 3000 and Biacore X100 biosensors has shown that all the proteins form molecular complexes with immobilized beta-amyloid peptide. The Kd values for these complexes varied from 8.36х10-8 M (peroxiredoxin) to 1.97х10-6 M (alpha-enolase). Oxidative modification of investigated proteins caused opposite effects on complexes of these peptides with beta-amyloid. The endogenous neuroprotector isatin increased dissociation of complexes formed by beta-amyloid peptide with both intact proteins (peroxiredoxin, glyceraldehyde-3-phosphate dehydrogenase) and/or oxidized proteins (peroxiredoxin, pyruvate kinase) used in this study.

Amyloid-binding proteins: affinity-based separation, proteomic identification, and optical biosensor validation.

The amyloid-beta peptide is considered as a key player in the development and progression of Alzheimer's disease (AD). Although good evidence exists that amyloid-beta accumulates inside cells, intracellular brain amyloid-beta-binding proteins remain poorly characterized. Here we describe a protocol for affinity-based profiling of amyloid-beta-binding proteins of rat brain, their proteomic identification and validation by a surface plasmon resonance (SPR)-based analysis. It includes: (a) SPR-based selection of immobilization conditions for beta-amyloid coupling and choice of appropriate resin for preparation of an affinity sorbent; (b) immobilization of beta-amyloid on the selected resin;

The effects of endogenous non-peptide molecule isatin and hydrogen peroxide on proteomic profiling of rat brain amyloid-β binding proteins: relevance to Alzheimer's disease?

The amyloid-β peptide is considered as a key player in the development and progression of Alzheimer’s disease (AD). Although good evidence exists that amyloid-β accumulates inside cells, intracellular brain amyloid-binding proteins remain poorly characterized. Proteomic profiling of rat brain homogenates, performed in this study, resulted in identification of 89 individual intracellular amyloid-binding proteins, and approximately 25% of them were proteins that we had previously identified as specifically binding to isatin, an endogenous neuroprotector molecule. A significant proportion of the amyloid-binding proteins (more than 30%) are differentially expressed or altered/oxidatively modified in AD patients. Incubation of brain homogenates with 70 µM hydrogen peroxide significantly influenced the profile of amyloid-β binding proteins and 0.1 mM isatin decreased the number of identified amyloid-β binding proteins both in control and hydrogen peroxide treated brain homogenates. The effects of hydrogen peroxide and isatin have been confirmed in optical biosensor experiments with purified glyceraldehyde-3-phosphate dehydrogenase, one of the known crucial amyloid-β binding proteins (also identified in this study). Data obtained suggest that isatin protects crucial intracellular protein targets against amyloid binding, and possibly favors intracellular degradation of this protein via preventing formation of amyloid-β oligomers described in the literature for some isatin derivatives.

Benzothiazole Aniline Tetra(ethylene glycol) and 3-Amino-1,2,4-triazole Inhibit Neuroprotection against Amyloid Peptides by Catalase Overexpression in Vitro

Alzheimer's disease, Familial British dementia, Familial Danish dementia, Type 2 diabetes mellitus, plus Creutzfeldt-Jakob disease are associated with amyloid fibril deposition and oxidative stress. The antioxidant enzyme catalase is a neuroprotective amyloid binding protein. Herein the effects of catalase overexpression in SH-SY5Y neuronal cells on the toxicity of amyloid-β (Aβ), amyloid-Bri (ABri), amyloid-Dan (ADan), amylin (IAPP), and prion protein (PrP) peptides were determined. Results showed catalase overexpression was neuroprotective against Aβ, ABri, ADan, IAPP, and PrP peptides. The catalase inhibitor 3-amino-1,2,4-triazole (3-AT) and catalase-amyloid interaction inhibitor benzothiazole aniline tetra(ethylene glycol) (BTA-EG4) significantly enhanced neurotoxicity of amyloid peptides in catalase overexpressing neuronal cells. This suggests catalase neuroprotection involves breakdown of hydrogen peroxide (H2O2) plus a direct binding interaction between catalase and the Aβ, ABri, ADan, IAPP, and PrP peptides. Kisspeptin 45-50 had additive neuroprotective actions against the Aβ peptide in catalase overexpressing cells. The effects of 3-AT had an intracellular site of action, while catalase-amyloid interactions had an extracellular component. These results suggest that the 3-AT and BTA-EG4 compounds may be able to inhibit endogenous catalase mediated neuroprotection. Use of BTA-EG4, or compounds that inhibit catalase binding to amyloid peptides, as potential therapeutics for Neurodegenerative diseases may therefore result in unwanted effects.

Gelsolin, a beta amyloid-binding protein is proteolytically cleaved in the brains of Alzheimer's disease and in adults with down syndrome

The deposition of fibrillar amyloid beta-protein (Aβ) is one of the main events in the amyloid plaques formation in Alzheimer's disease (AD) and adult Down syndrome (DS) individuals. Accumulating evidence from our and other groups has shown that gelsolin, a cytoplasmic and secretory protein, regulates the fibrillogenesis of Aβ. Gelsolin is constitutively expressed through out the central nervous system, and it inhibits the fibrillization of Aβ, and defibrillizes the preformed Aβ fibrils. We studied whether there is a relationship between the levels of Aβ and gelsolin in the brains of AD and DS subjects. The homogenates of postmortem brain samples of frontal cortex from patients with AD (age 64-85 y, n = 8) and DS (age 43-63 y) and age-matched control subjects were prepared. Then homogenates were analyzed for gelsolin and its proteolytic fragment by Western blot. The levels of Aβ were measured in the homogenates by ELISA.. Our results showed the presence of a carboxyl-terminal gelsolin peptide (CTF-gelsolin), a proteolytically cleaved fragment of gelsolin, in the brain sample from AD but not in age-matched controls (n = 6). A correlation was observed between the levels of CTF-gelsolin and severity of the disease. Similarly, we observed that proteolysis of gelsolin also occurs in the brains of adult DS (n = 7) but not in age-matched control (n = 6) subjects. The levels of soluble Aβ40/Aβ42 were increased in the brains of adult patients with DS. A positive correlation was observed between the levels of soluble Aβ40/Aβ 42, and the proteolysis of gelsolin. We suggest that gelsolin cleavage in the brains may act as a marker of amyloid -mediated pathogenesis in AD and DS.

The endogenous peptides of normal human serum extracted from the acetonitrile-insoluble precipitate using modified aqueous buffer with analysis by LC–ESI–Paul ion trap and Qq-TOF

Many peptides of biological or medicinal importance may be derived from proteolytic actions and are found at low concentrations in human blood fluids. Endogenous polypeptides from human serum were precipitated in acetonitrile and the precipitate was then selectively extracted with water modified by organic solvents and collected over C18 resin. Extraction of serum with C18 alone, and the acetonitrile supernatant or ultrafiltration collected over C18, served as controls. The samples were analyzed by SDS-PAGE, or C18 high pressure liquid chromatography with electrospray ionization using a Paul ion trap and Qq-TOF. Spectra were correlated without specifying an enzyme using the X!TANDEM or the Paragon algorithms. Multiple endogenous peptides from plasminogen, coagulation factors, collagens, serum amyloid, receptors, zinc finger/bromo peptide proteins, ryanodine receptor, calmodulin binding activator, erythroid differentiation factor, testes cancer antigen, extracellular matrix protein, myeloid/lymphoid leukemia 2 and many low abundance proteins were correlated by X!TANDEM with protein expect values of approximately E-16 or less. Proteins with binding sites for nucleic acids, phosphoinositides, and other cellular locations were also observed using the Qq-TOF and Paragon algorithm. Proteins with low expectation scores and overlapping peptides sequences were observed. The existence of these proteins in serum has been confirmed by tryptic digestion and LC-ESI-MS/MS. The presence of plasminogen, serum amyloid and zinc finger RNA binding proteins were confirmed by Western blot. There was agreement on the detection of endogenous peptides from low abundance proteins associated with the biology of cancer from the examination of the blood peptides by ion trap and Qq-TOF, tryptic digests of blood proteins, and Western blot.

Peripheral Transgene Expression of Plasma Gelsolin Reduces Amyloid in Transgenic Mouse Models of Alzheimer's Disease

The accumulation and deposition of the 40-42-amino acid peptide amyloid beta (Abeta) is thought to be a critical event in the pathology of Alzheimer's disease (AD). Both passive and active immunizations against Abeta in amyloid-depositing transgenic mice have reduced Abeta pathology and improved memory-related behavior. Peripheral treatments with other amyloid-binding agents have also reduced Abeta pathology. The present study demonstrates that peripheral delivery of plasmid DNA coding for the amyloid-binding protein plasma gelsolin reduces brain Abeta in two separate amyloid-depositing transgenic mouse models of AD when inter-litter variability is accounted for. The reduction in Abeta pathology observed is accompanied by an apparent increase in activated and reactive microglia and soluble oligomeric forms of amyloid. These findings demonstrate that peripheral expression of plasma gelsolin may be a suitable gene-therapeutic approach for the prevention or treatment of AD.

High density lipoproteins bind Aβ and apolipoprotein C-II amyloid fibrils

Disease-associated amyloid deposits contain both fibrillar and nonfibrillar components. The majority of these amyloid components originate or coexist in the bloodstream. To understand the nature of the interaction between the nonfibrillar and fibrillar components, we have developed a centrifugation method to isolate fibril binding proteins from human serum. Amyloid fibrils composed of either Abeta peptide or apolipoprotein C-II (apoC-II) cosedimented with specific serum proteins. Gel electrophoresis, mass spectrometry peptide fingerprinting, and Western analysis identified the major binding species as proteins found in HDL particles, including apoA-I, apoA-II, apoE, clusterin, and serum amyloid A. Sedimentation analysis showed that purified human HDL and recombinant apoA-I lipid particles bound directly to Abeta and apoC-II amyloid fibrils. These studies reveal a novel function of HDL that may contribute to the well-established protective effect of this lipoprotein class in heart disease.

Differentiation-dependent expression of 17beta-hydroxysteroid dehydrogenase, type 10, in the rodent testis: effect of aging in Leydig cells.

Expression of the new 17beta-hydroxysteroid dehydrogenase (HSD), type 10 (17beta-HSD-10), formerly known as endoplasmic reticulum-associated amyloid-binding protein, has been investigated in the testes of various mammals under normal and perturbed conditions. Results show that 17beta-HSD-10 is a major product of both fetal and adult-type Leydig cells. In the former, protein persists until late in postnatal development; and in the short-day hamster model, it does not disappear when Leydig cells involute. During puberty in the rat, immunohistochemical staining for 17beta-HSD-10 in adult-type Leydig cells first becomes evident on d 20, increasing to maximal staining intensity by d 35. In the rat, but not in the mouse or any other species examined, there is also staining in late spermatids. Examination of testes from rats subjected to perinatal treatment with either a GnRH antagonist or low and high doses of diethylstilbestrol revealed that expression of 17beta-HSD-10 follows closely Leydig cell differentiation status, correlating with 3beta-HSD expression in a previous study. In aging (23 months) rat testes, Leydig cell (but not germ cell) immunostaining for 17beta-HSD-10 is markedly reduced. 17beta-HSD-10 seems to preferentially convert 3alpha-androstanediol into dihydrotestosterone, and estradiol to estrone. Thus, perinatal expression of this enzyme in fetal Leydig cells may contribute to protecting these cells from estrogens and encourage androgen formation.

Influence of double (APP and PS1) mutations on sprouting in the hippocampus following entorhinal cortex lesions in mice and the effects of estrogen

In Alzheimer's disease, the main component of amyloid deposits is a 39–43 amino acid peptide referred to as amyloid peptide or A β. A crucial issue in the study of this disorder is to define the sequence of events that lead to amyloid deposition. In the present study, a new approach was developed that allows to specifically solubilize A β peptide trapped within amyloid deposits and to quantify its amount by dot-blot immunoassay. The present method also permits to isolate components tightly bound to A β and that are likely to catalyze its aggregation. Biochemical A β quantitation was performed in 4 Brodmann areas from 17 elderly individuals exhibiting different degrees of amyloidosis. In parallel, classical neuropathology was done by histochemical and immunohistochemical methods. A β amounts (pmol) were correlated to the number of amyloid deposits determined by neuropathology showing high statistical significance. Moreover, amyloid-binding proteins including apolipoprotein E and heparan sulfate proteoglycans were also found associated to A β in the amyloid preparation. The present biochemical procedure is a new and reliable method to quantify amyloid deposition in brain. Furthermore, it allows to detect amyloid-associated components such as apolipoprotein E, that may be involved in the pathological process of amyloidogenesis.

Alpha2-macroglobulin associates with beta-amyloid peptide and prevents fibril formation.

We have used the yeast two-hybrid system to isolate cDNAs encoding proteins that specifically interact with the 42-aa beta-amyloid peptide (Abeta), a major constituent of senile plaques in Alzheimer's disease. The carboxy terminus of alpha2-macroglobulin (alpha2M), a proteinase inhibitor released in response to inflammatory stimuli, was identified as a strong and specific interactor of Abeta, utilizing this system. Direct evidence for this interaction was obtained by co-immunoprecipitation of alpha2M with Abeta from the yeast cell, and by formation of SDS-resistant Abeta complexes in polyacrylamide gels by using synthetic Abeta and purified alpha2M. The association of Abeta with alpha2M and various purified amyloid binding proteins was assessed by employing a method measuring protein-protein interactions in liquid phase. The dissociation constant by this technique for the alpha2M-Abeta association using labeled purified proteins was measured (Kd = 350 nM). Electron microscopy showed that a 1:8 ratio of alpha2M to Abeta prevented fibril formation in solution; the same ratio to Abeta of another acute phase protein, alpha1-antichymotrypsin, was not active in preventing fibril formation in vitro. These results were corroborated by data obtained from an in vitro aggregation assay employing Thioflavine T. The interaction of alpha2M with Abeta suggests new pathway(s) for the clearance of the soluble amyloid peptide.

Alzheimer neurofibrillary lesions: Molecular nature and potential roles of different components

Neurofibrillary lesions found in Alzheimer disease (AD) are known to react with antibodies raised against different molecules. At least 20 components have been detected in neurofibrillary tangles. These components can be roughly categorized into five groups, which include structural proteins, kinases and other cytosolic enzymes, stress-related molecules, amyloid and amyloid binding proteins, and others. Among them, an abnormal form of microtubule associated protein tau, PHF-tau, is a major component of Alzheimer NFT. Kinases associated with NFT, especially those belonging to the family of proline-directed Ser/Thr kinases, are considered to be important for PHF-tau hyperphosphorylation. A potentially significant kinase is a Cdc2-related kinase, which is associated tightly with paired helical filaments, has a molecular weight of 33kDa and is different from other known Cdc2-related kinases. The possibility that some of the NFT-associated elements may play an active role in the pathogenesis of Alzheimer's disease was supported by recent studies, in which advanced glycated products and markers of oxidant stress were located in NFT. In addition, PHF-tau was found to be glycated, and in vitro glycated tau was capable of inducing oxidant stress. Further characterization of different components of NFT by biochemical and other approaches will be important for understanding the mechanisms involved in the supramolecular aggregation of PHF within NFT.