Paired Helical Filament Tau Research Articles

Formation of Tau Inclusions in Knock-in Mice with Familial Alzheimer Disease (FAD) Mutation of Presenilin 1 (PS1)

Mutations in the presenilin 1 (PS1) gene are responsible for the early onset of familial Alzheimer disease (FAD). Accumulating evidence shows that PS1 is involved in gamma-secretase activity and that FAD-associated mutations of PS1 commonly accelerate Abeta(1-42) production, which causes Alzheimer disease (AD). Recent studies suggest, however, that PS1 is involved not only in Abeta production but also in other processes that lead to neurodegeneration. To better understand the causes of neurodegeneration linked to the PS1 mutation, we analyzed the development of tau pathology, another key feature of AD, in PS1 knock-in mice. Hippocampal samples taken from FAD mutant (I213T) PS1 knock-in mice contained hyperphosphorylated tau that reacted with various phosphodependent tau antibodies and with Alz50, which recognizes the conformational change of PHF tau. Some neurons exhibited Congo red birefringence and Thioflavin T reactivity, both of which are histological criteria for neurofibrillary tangles (NFTs). Biochemical analysis of the samples revealed SDS-insoluble tau, which under electron microscopy examination, resembled tau fibrils. These results indicate that our mutant PS1 knock-in mice exhibited NFT-like tau pathology in the absence of Abeta deposition, suggesting that PS1 mutations contribute to the onset of AD not only by enhancing Abeta(1-42) production but by also accelerating the formation and accumulation of filamentous tau.

Possible Role of Each Repeat Structure of the Microtubule-Binding Domain of the Tau Protein in In Vitro Aggregation

Although one of the priorities in Alzheimer's research is to clarify the filament formation mechanism of the tau protein, it is currently unclear how it is transformed from a normal structure in a neuron. To examine which part and what structural change in the tau protein are involved in its transformation into a pathological entity, the initial in vitro self-aggregation features of each repeat peptide (R1-R4) constituting a three- or four-repeat microtubule-binding domain (3RMBD or 4RMBD) in the tau protein was investigated by measuring both the fluorescence and light scattering (LS) spectra on the same instrument, because these MBD domains constitute the core moiety of the tau paired helical filament (PHF) structure. The conformational features of the R1 and R4 peptides in trifluoroethanol were also investigated by (1)H-NMR and molecular modeling analyses and compared with those of the R2 and R3 peptides. The analyses of the LS spectra clarified (i) the self-aggregation rates of R1-R4, 3RMBD and 4RMBD at a fixed concentration (15 mM), (ii) their minimum concentrations for starting filament extension, and (iii) the concentration dependence of their self-aggregations. The fluorescence analyses showed that the R2 and R3 peptides have high self-aggregation abilities at the extension and nucleation steps, respectively, in their filament formation processes. It was shown that the R2 repeat exhibits a positive synergistic effect on the aggregation of 4RMBD. The R1 and R4 repeats, despite their weak self-aggregation abilities, are necessary for the intact PHF formation of tau MBD, whereas they exerted a negative effect on the R3-driven aggregation of 3RMBD. The conformational analyses showed the importance of the amphipathic conformational features of the R1 to R4 peptides, and the intermolecular disulfide bonding abilities of the R2 and R3 peptides for the PHF formation. On the basis of the present spectral and conformational results, the possible role of each repeat structure in the dimeric formation of MBD at the initial in vitro aggregation stage is discussed.

Lithium Chloride Increases the Production of Amyloid-β Peptide Independently from Its Inhibition of Glycogen Synthase Kinase 3

Glycogen synthase kinase 3 (GSK3) is able to phosphorylate tau at many sites that are found to be phosphorylated in paired helical filaments in Alzheimer disease. Lithium chloride (LiCl) efficiently inhibits GSK3 and was recently reported to also decrease the production of amyloid-beta peptide (Abeta) from its precursor, the amyloid precursor protein. Therefore, lithium has been proposed as a combined therapeutic agent, inhibiting both the hyperphosphorylation of tau and the production of Abeta. Here, we demonstrate that the inhibition of GSK3 by LiCl induced the nuclear translocation of beta-catenin in Chinese hamster ovary cells and rat cultured neurons, in which a decrease in tau phosphorylation was observed. In both cellular models, a nontoxic concentration of LiCl increased the production of Abeta by increasing the beta-cleavage of amyloid precursor protein, generating more substrate for an unmodified gamma-secretase activity. SB415286, another GSK3 inhibitor, induced the nuclear translocation of beta-catenin and slightly decreased Abeta production. It is concluded that the LiCl-mediated increase in Abeta production is not related to GSK3 inhibition.

Visualization of Newly Deposited tau in Neurofibrillary Tangles and Neuropil Threads

Neurofibrillary tangles (NFTs) and neuropil threads (NTs), the major hallmark of Alzheimer disease (AD), are composed of the microtubule-associated protein tau that has undergone posttranslational modifications, including deamidation and isomerization on asparaginyl or aspartyl residues. Because such modifications represent protein aging, we generated 2 antibodies, TM4, specific for Asp-387 of tau, and iD387, specific for isoAsp-387 of tau, to investigate the evolution of NFTs and NTs. On Western blots of Sarkosyl-insoluble fractions, TM4 strongly labeled paired helical filament-tau (PHF-tau), whereas iD387 preferentially labeled PHF smear. Thus, it is reasonable to postulate that TM4-labeled tau (unmodified tau species) represents more recent deposition, and iD387-labeled tau (modified tau species) represents earlier deposition. Unexpectedly, TM4 immunostained even highly evolved NFTs, suggesting that deposition of newly produced tau continues until neuronal death. iD387 labeled the whole profile of NFTs up to distal dendritic branches, whereas TM4 staining was localized to particular portions of NFTs in proximal dendrites and neuronal perikarya. In NTs, TM4 preferentially labeled the outer portion, whereas iD387 intensely labeled the core portion. Based on TM4-positive NFT counts and total NFT counts, we speculate that NFTs in the human hippocampus are produced at a constant rate irrespective of the disease stage.

Sites of Tau Important for Aggregation Populate β-Structure and Bind to Microtubules and Polyanions

The aggregation of the microtubule-associated tau protein and formation of "neurofibrillary tangles" is one of the hallmarks of Alzheimer disease. The mechanisms underlying the structural transition of innocuous, natively unfolded tau to neurotoxic forms and the detailed mechanisms of binding to microtubules are largely unknown. Here we report the high-resolution characterization of the repeat domain of soluble tau using multidimensional NMR spectroscopy. NMR secondary chemical shifts detect residual beta-structure for 8-10 residues at the beginning of repeats R2-R4. These regions correspond to sequence motifs known to form the core of the cross-beta-structure of tau-paired helical filaments. Chemical shift perturbation studies show that polyanions, which promote paired helical filament aggregation, as well as microtubules interact with tau through positive charges near the ends of the repeats and through the beta-forming motifs at the beginning of repeats 2 and 3. The high degree of similarity between the binding of polyanions and microtubules supports the hypothesis that stable microtubules prevent paired helical filament formation by blocking the tau-polyanion interaction sites, which are crucial for paired helical filament formation.

Proteomic analysis of neurofibrillary tangles in Alzheimer disease identifies GAPDH as a detergent‐insoluble paired helical filament tau binding protein

We performed proteomic analysis of neurofibrillary tangles (NFTs) obtained by laser capture microdissection from pyramidal neurons in hippocampal sector CA1 in patients with Alzheimer disease (AD) using liquid chromatography (LC)-mass spectrometry (MS)/MS. We discovered a total of 155 proteins in laser captured NFT's, 72 of which were identified by multiple unique peptides. Of these 72 proteins, 63 had previously unknown association with NFTs; one of these was glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We validated by immunohistochemistry that GAPDH co-localized with the majority of NFTs as well as plaque-like structures in AD brain and was co-immunoprecipitated by antibodies to abnormal forms of tau in AD, but not tau from AD temporal cortex. Characterization of GAPDH showed that it, along with phosphorylated tau and Abeta peptides, was present in detergent-insoluble fractions from AD temporal cortex but not from age-matched controls. These data are the first proteomic investigation of NFTs. Moreover, our results validate this approach by demonstrating that GAPDH, a glycolytic and microtubule binding protein, not only co-localized to NFTs and immunoprecipitated with PHF-tau, but also is one of the few proteins known to undergo conversion to a detergent-insoluble form in AD.

Proteolysis of Non-phosphorylated and Phosphorylated Tau by Thrombin

The microtubule-associated protein tau aggregates intracellularly by unknown mechanisms in Alzheimer's disease and other tauopathies. A contributing factor may be a failure to break down free cytosolic tau, thus creating a surplus for aggregation, although the proteases that degrade tau in brain remain unknown. To address this issue, we prepared cytosolic fractions from five normal human brains and from perfused rat brains and incubated them with or without protease inhibitors. D-Phenylalanyl-L-prolylarginyl chloromethyl ketone, a thrombin-specific inhibitor, prevented tau breakdown in these fractions, suggesting that thrombin is a brain protease that processes tau. We next exposed human recombinant tau to purified human thrombin and analyzed the fragments by N-terminal sequencing. We found that thrombin proteolyzed tau at multiple arginine and lysine sites. These include Arg(155)-Gly(156), Arg(209)-Ser(210), Arg(230)-Thr(231), Lys(257)-Ser(258), and Lys(340)-Ser(341) (numbering according to the longest human tau isoform). Temporally, the initial cleavage occurred at the Arg(155)-Gly(156) bond. Proteolysis of the resultant C-terminal tau fragment then proceeded bidirectionally. When tau was phosphorylated by glycogen synthase kinase-3beta, most of these proteolytic processes were inhibited, except for the first cleavage at the Arg(155)-Gly(156) bond. Furthermore, paired helical filament tau prepared from Alzheimer's disease brain was more resistant to thrombin proteolysis than following dephosphorylation by alkaline phosphatase. The results suggest a possible role for thrombin in proteolysis of tau under physiological and/or pathological conditions in human brains. They are consistent with the hypothesis that phosphorylation of tau inhibits proteolysis by thrombin or other endogenous proteases, leading to aggregation of tau into insoluble fibrils.

Structural evaluation of conformational transition state responsible for self-assembly of tau microtubule-binding domain

In the brains of Alzheimer’s disease patients, the tau protein abnormally aggregates to form an insoluble paired helical filament (PHF). Since the third repeat structure (R3) of the tau microtubule-binding domain plays an essential role in PHF formation and self-aggregates most significantly in an aqueous solution of 20–40% trifluoroethanol (TFE), its possible conformation was estimated from the combination of (i) the TFE-dependent deviations of NH and CαH proton chemical shifts from those of the random structure in water and (ii) the TFE-dependent NOE effect connectivity diagrams between the neighboring protons. Consequently, it was indicated that the extended structure of the N-terminal VQIVYK moiety and the α-helical-like structure of the LSKVTSKC region provide a structural scaffold for initiating the self-assembled filament formation of the R3 structure. To the best of our knowledge, this is the first study that demonstrated the initial structural moiety and its structural feature necessary for starting the tau PHF formation.

14-3-3 proteins and zeta isoform containing neurofibrillary tangles in patients with Alzheimer's disease.

Immunolocalization of 14-3-3 proteins in Alzheimer's disease (AD) brains was investigated using isoform-specific antibodies. Weak granular immunoreactivity of 14-3-3 proteins was found in neuronal cytoplasm in control subjects and AD brains. Both intracellular and extracellular neurofibrillary tangles (NFTs), as well as neuropil thread-like structures, were immunopositive for 14-3-3 proteins. This was corroborated by triple-fluorolabeling method visualizing paired helical filament (PHF) tau and 14-3-3 epitopes in relation to fibrillary state detected by thiazin red. Pretangle neurons (positive for PHF-tau without fibrillary structure detected by thiazin red) only contained fine granular immunoreactivity (IR) of 14-3-3, which was similarly found in unaffected neurons. Granular cytoplasmic IR of 14-3-3 proteins in pretangle neurons was not colocalized to granular tau-like IR, which suggests that participation of 14-3-3 proteins in NFT formation was restricted to its later stages. Its zeta isoform was most prominent in these NFTs, suggesting that this isoform is a major component involved in the formation of NFTs. In contrast, IR of epsilon isoform was found in the neuropil of the hippocampus and that of sigma isoform was localized to granule cells of the dentate gyrus in AD brains, as seen in the age-matched controls. Expression of 14-3-3 proteins were found to be highly variable and dependent on their isoforms, regions and cell types. Molecular, as well as topographical, dissection of 14-3-3 proteins will provide us with an improved understanding of this molecule in normal and pathological conditions.

Folding of Alzheimer's core PHF subunit revealed by monoclonal antibody 423

At present, the conformation-dependent monoclonal antibodies (mAb) provide the only information on folding of tau in the core PHF. Monoclonal antibody MN423 recognizes all and only those Alzheimer's disease (AD) core paired helical filaments (PHFs) subunits, which terminate at Glu391. Using recombinant analogs of the core PHF subunit corresponding to tau residues τ297–391, we found that the C-terminal pentapeptide 387DHGAE 391 represented only one component of the structure recognized by mAb 423. Therefore, deletion mutants of the core subunit were generated to identify assembled parts of this conformational structure. We localized two spatially close components in the region 306–325 ( 306VQIVYK 311 and 321KCGSL 325) contributing to formation of the structure identified by mAb 423. Thus, the spatial proximity of three subunit segments 306VQIVYK 311, 321KCGSL 325 and 387DHGAE 391 represents constraints for intramolecular folding of the core PHF subunit. Since PHF represents a compelling drug target in AD, structural knowledge presented could contribute to structure-based drug design.

Glycogen synthase kinase 3: a drug target for CNS therapies.

Abstract Glycogen synthase kinase3 (GSK3) is emerging as a prominent drug target in the CNS. The most exciting of the possibilities of GSK3 lies within the treatment of Alzheimer's disease (AD) where abnormal increases in GSK3 levels and activity have been associated with neuronal death, paired helical filament tau formation and neurite retraction as well as a decline in cognitive performance. Abnormal activity of GSK3 is also implicated in stroke. Lithium, a widely used drug for affective disorders, inhibits GSK3 at therapeutically relevant concentrations. Thus while the rationale remains testable, pharmaceutical companies are investing in finding a selective inhibitor of GSK3. In the present review, we summarize the properties of GSK3, and discuss the potential for such a therapy in AD, and other CNS disorders.

Binding of Tau to Heat Shock Protein 27 Leads to Decreased Concentration of Hyperphosphorylated Tau and Enhanced Cell Survival

Pathological hyperphosphorylated tau is the principal component of paired helical filaments, a pathological hallmark of Alzheimer disease (AD) and a strong candidate for a neurotoxic role in AD and other neurodegenerative disorders. Here we show that heat shock protein 27 (Hsp27) preferentially binds pathological hyperphosphorylated tau and paired helical filaments tau directly but not non-phosphorylated tau. The formation of this complex altered the conformation of pathological hyperphosphorylated tau and reduced its concentration by facilitating its degradation and dephosphorylation. Moreover, Hsp27 rescues pathological hyperphosphorylated tau-mediated cell death. Therefore, Hsp27 is likely to provide a neuroprotective effect in AD and other tauopathies.

Biochemical and ultrastructural study of neurofibrillary tangles in amyotrophic lateral sclerosis/parkinsonism-dementia complex in the Kii peninsula of Japan.

Amyotrophic lateral sclerosis/parkinsonism-dementia complex of the Kii peninsula (Kii ALS/PDC) is a neurodegenerative disorder endemic to natives in the southern coast area of the Kii peninsula of Japan. The disorder closely resembles Guamanian ALS/PDC clinically and neuropathologically. The characteristic neuropathological finding is abundant neurofibrillary tangles (NFTs) without amyloid deposition. To elucidate the biochemical properties of hyperphosphorylated tau protein, the major component of the NFTs, we examined Kii ALS/PDC brains by immunoblotting and immunohistochemical analysis using well-characterized anti-tau antibodies specific to phosphorylation-dependent or -independent epitopes. Hyperphosphorylated tau in Kii ALS/PDC had phosphorylated epitopes common to tau of paired helical filaments (PHFs) in Alzheimer disease (AD): immunoblot showed triplet bands composed of 6 tau isoforms. Ultrastructurally, NFTs revealed a twisted filamentous shape similar to PHF of AD. The biochemical properties of its phosphorylated tau protein and the ultrastructural characteristics of the NFTs of Kii ALS/PDC are very similar, if not identical, to PHF tau in AD, although they are different taupopathies.

Brain biopsy prior to treatment of Alzheimer's disease.

A cortical biopsy was analyzed using immunohistochemical methodology in a total of 213 patients with suspected normal pressure hydrocephalus (NPH). Normal intracranial pressure (ICP) was registered in 121 (43 %) of patients with suspected NPH. The incidence of Alzheimer's disease (AD) related lesions was quite high, since 38 % of all subjects displayed beta-amyloid (betaA4) aggregates and in 8 % of subjects paired helical filament-tau (PHF-tau) pathology was noted. The AD associated pathologies were more common in subjects with normal than elevated ICP. Of the 121 subjects with NPH, 16 (13 %) had subclinical, histopathologically verified AD and a further 40 subjects (33 %) could be considered to be at high risk to develop AD. Pharmacological treatment trials of AD should be carried out on subjects with evident brain pathology (betaA4 aggregates or PHF-tau pathology) especially during the early stage of the disease, i.e., the subclinical stage. For this purpose a simple cortical biopsy with a low risk of complication would represent a diagnostic "method" of choice.

Seladin-1 transcription is linked to neuronal degeneration in Alzheimer’s disease

Seladin-1 is a gene recently shown to be down-regulated in brain regions selectively degenerated in Alzheimer’s disease. The sequence of seladin-1 shares similarities with flavin-adenine-dinucleotide-dependent oxidoreductases and it has been found to protect cells from apoptotic cell death. In this work, we show that the transcription of seladin-1 is selectively down-regulated in the brain areas affected in Alzheimer’s disease. The down-regulation in seladin-1 transcription was associated with hyperphosphorylated tau seen as linkage to immunohistochemically detected paired helical filament tau, neuritic plaques and neurofibrillary tangles. In contrast, no association was found between seladin-1 transcription and β-amyloid deposition when analyzing human samples or tissue from transgenic animals. Furthermore, the relative transcription of seladin-1 was found to fluctuate during aging in the transgenic mouse model of Alzheimer’s disease. The fluctuation was enhanced by Alzheimer’s disease causing mutations in presenilin-1 and amyloid precursor protein genes. Finally, seladin-1 transcription was found to be up-regulated in mouse N2a cells induced to undergo apoptosis with okadaic acid. The results presented here indicate that seladin-1 transcription is selectively down-regulated in brain regions vulnerable to Alzheimer’s disease and this down-regulation is associated with the hyperphosphorylation of tau protein.

Pathological Determinants of the Transition to Clinical Dementia in Alzheimer's Disease

The authors developed multivariate models to examine the association between Clinical Dementia Rating (CDR) scale scores and the spatial distribution of paired helical filament tau (PHF-tau) pathology. Severity of tauopathy was examined in 14 cortical regions of interest (ROIs). Classification trees were used to test the independent hierarchical contributions of each ROI to dementia. Multiple-regression and cluster analyses were performed to determine the relative contributions of select ROIs to dementia. Dementia (CDR S 1) was modeled as a dichotomous variable. Autopsy material was obtained from 124 demented and nondemented elderly patients. All ROIs except the hippocampus made significant contributions to dementia. However, they were not independent. In multivariate models, only a single step (Step 7 in a hierarchical progression), which contained four ROIs, contributed significantly to dementia. A classification tree resulted in a single decision split, suggesting that only Step 7 ROIs need be considered. A total of 89.4% of the cases were correctly classified (p < .0001). Twelve discrepant cases all had superimposed vasculopathy that might also have affected the function of Step 7 ROIs. The transition to clinical dementia was associated with the presence of tauopathy in A9/10, A22, A23, and A39. Animal studies suggest that these represent a single distributed cortical network focusing on prefrontal regions that provide "executive control" over complex goal-directed behaviors. A22, A23, and A39 provide major afferents to other frontal systems and have previously been implicated in very early clinical Alzheimer's disease.

Transglutaminase bonds in neurofibrillary tangles and paired helical filament tau early in Alzheimer's disease

Transglutaminase-catalyzed ε(γ-glutamyl)lysine cross-links exist in Alzheimer's disease (AD) paired helical filament (PHF) tau protein but not normal soluble tau. To test the hypothesis that these cross-links could play a role in the formation of neurofibrillary tangles (NFT), we used single- and double-label immunofluorescence confocal microscopy and immunoaffinity purification and immunoblotting to examine ε(γ-glutamyl)lysine cross-links in AD and control brains. The number of neurons that are immunoreactive with an antibody directed at the ε-(γ-glutamyl)lysine bond was significantly higher in AD cortex compared with age-matched controls and schizophrenics. PHF tau-directed antibodies AT8, MC-1 and PHF-1 co-localized with ε(γ-glutamyl)lysine immunolabeling in AD NFT. Immunoaffinity purification and immunoblotting experiments demonstrated that PHF tau contains ε(γ-glutamyl)lysine bonds in parietal and frontal cortex in AD. In control cases with NFT present in the entorhinal cortex and hippocampus, indicative of Braak and Braak stage II, ε(γ-glutamyl)lysine bonds were present in PHF tau in parietal and frontal cortex, despite the lack of microscopically detectable NFT or senile plaques in these cortical regions. The presence of PHF tau with ε(γ-glutamyl)lysine bonds in brain regions devoid of NFT in stage II (but regions, which would be expected to contain NFT in stage III) suggests that these bonds occur early in the formation of NFT.

Cotton wool plaques in non-familial late-onset Alzheimer disease.

Cotton wool plaques (CWP) are large, ball-like plaques lacking dense amyloid cores that displace adjacent structures. They were first described in a Finnish kindred with early-onset Alzheimer disease (AD) with spastic paraparesis due to a presenilin-1 delta9 mutation. We describe a case of sporadic late-onset AD with numerous neocortical CWP as well as severe amyloid angiopathy and marked leukoencephalopathy, compared with 16 cases of late-onset AD with similar degrees of amyloid angiopathy and leukoencephalopathy. The cases were studied with histologic methods and with single and double immunostaining for beta-amyloid (Abeta), paired helical filaments-tau (PHF-tau), neurofilament (NF), glial fibrillary acidic protein (GFAP), HLA-DR, and amyloid precursor protein (APP). We found that CWP were well-circumscribed amyloid deposits infiltrated by ramified microglia and surrounded by dystrophic neurites that were immunopositive for APP, but only weakly for NF and PHF-tau. Abeta1-12 was diffuse throughout the CWP, while Abeta37-42 was peripherally located and Abeta20-40 more centrally located. Two of the 16 late-onset AD cases also had CWP, but they were also admixed with diffuse plaques and plaques with dense amyloid cores. Pyramidal tract degeneration was not a consistent finding or a prominent feature in any case. The results suggest that CWP are not specific for early-onset familial AD with spastic paraparesis.

Molecular profiling of paired helical filaments

Fibrous deposition of neurofibrillary tangles (NFT) is one of the neuropathological hallmarks of Alzheimer’s disease (AD). The extent of the NFT lesions correlates well with the severity of dementia in AD. Therefore, it is of ultimate importance to understand the molecular basis of NFT. This would entails a formidable challenge that requires the isolation of a defined macromolecular assembly and characterization of individual constitutes by conventional biochemical analysis. Fortunately, the first step has been largely aided by the ultrastructural studies using electron microscopy that led to the identification of the major fibrous units of NFT as paired helical filaments (PHF) [1,2]. In the recent decade, major efforts and significant progress have been made in unraveling the molecular composition of PHF. However, as the article by Goux et al. (this issue) and other studies suggest, such an endeavor is far from over. Goux et al. argue that the current view of PHF as made mainly of tau protein may only represent a partial picture of molecules that are present in PHFs. Based on their analysis; they maintained that other non-protein components such as glycolipids might be more abundant than tau on a weight percent basis. While the view of Goux et al. is likely to be controversial, the involvement of molecules other than tau protein in the composition of or the assembly of PHF has been suggested by other investigators. In addition to glycolipid, ubiquitin [3], glycosaminoglycans [4,5], products of glycation and lipid oxidation [6], and metals [7,8] have been found to be associated with PHF. Like any other biological assembly, the key problem in differentiating authentic PHF components from “contaminants” is how PHF structures should be defined, analyzed and quantitated. Electron microscopy (EM) continues to be the standard method for verifying the presence of PHF through the observation of the characteristic, twisted fibrous structures. While it is possible to obtain estimates on mass per unit length of the PHF using scanning transmission EM (STEM) [2], other methods are necessary for analyzing the molecular nature and composition of PHF. The first insight into the major protein components came as antibodies against PHF cross-react with tau. After extensive analysis of isolated PHF, it is commonly accepted that hyperphosphorylated tau is a major protein component of PHF [9]. However, few studies have actually quantitated tau on a weight percent basis in their preparations. It turns out that quantitation of tau and other components in PHF is not as simple as it appears. Tau exists as six isoforms and is extensively modified by phosphorylation, and possibly by crosslinking. The endogenous proteolysis at different stages of AD pathology also results in tau molecules of various lengths. The insolubility of PHF also adds to the complexity in molecular analysis by conventional methods. PHF can be isolated as pronase-resistant tangle fragments (prcPHF) with truncated tau containing only the repeat region of 90 residues at the protein core. Since prcPHF is extremely insoluble and resists extraction by denaturants and detergents, there may be uncertainty in the purity analysis for the prcPHF preparations other than gross morphology examination by EM. It is in this type of preparations that Goux and co-workers initially find the evidence of glycolipids being abundant nonprotein components [10]. Using H and C NMR and GC/MS, they found evidence of carbohydrates (mostly glucose) and fatty acids with a molar ratio of about 6:1. Their data was then fit to a proposed structure of

Phosphorylation-mimicking glutamate clusters in the proline-rich region are sufficient to simulate the functional deficiencies of hyperphosphorylated tau protein.

The microtubule-associated tau proteins represent a family of closely related phosphoproteins that become enriched in the axons during brain development. In Alzheimer's disease (AD), tau aggregates somatodendritically in paired helical filaments in a hyperphosphorylated form. Most of the sites that are phosphorylated to a high extent in paired helical filament tau are clustered in the proline-rich region (P-region; residues 172--251) and the C-terminal tail region (C-region; residues 368--441) that flank tau's microtubule-binding repeats. This might point to a role of a region-specific phosphorylation cluster for the pathogenesis of AD. To determine the functional consequences of such modifications, mutated tau proteins were produced in which a P- or C-region-specific phosphorylation cluster was simulated by replacement of serine/threonine residues with glutamate. We show that a phosphorylation-mimicking glutamate cluster in the P-region is sufficient to block microtubule assembly and to inhibit tau's interaction with the dominant brain phosphatase protein phosphatase 2A isoform AB alpha C. P-region-specific mutations also decrease tau aggregation into filaments and decrease tau's process-inducing activity in a cellular transfection model. In contrast, a phosphorylation-mimicking glutamate cluster in the C-region is neutral with regard to these activities. A glutamate cluster in both the P- and C-regions induces the formation of SDS-resistant conformational domains in tau and suppresses tau's interaction with the neural membrane cortex. The results indicate that modifications in the proline-rich region are sufficient to induce the functional deficiencies of tau that have been observed in AD. They suggest that phosphorylation of the proline-rich region has a crucial role in mediating tau-related changes during disease.