EFhd2 is a novel amyloid protein associated with pathological tau in Alzheimer's disease

EFhd2 is a conserved calcium‐binding protein, abundant within the central nervous system. Previous studies identified EFhd2 associated with pathological forms of tau proteins in the tauopathy mouse model JNPL3, which expresses the human tau^P301L mutant. This association was validated in human tauopathies, such as Alzheimer's disease (AD). However, the role that EFhd2 may play in tauopathies is still unknown. Here, we show that EFhd2 formed amyloid structures in vitro, a capability that is reduced by calcium ions. Electron microscopy (EM) analyses demonstrated that recombinant EFhd2 formed filamentous structures. EM analyses of sarkosyl‐insoluble fractions derived from human AD brains also indicated that EFhd2 co‐localizes with aggregated tau proteins and formed granular structures. Immunohistological analyses of brain slices demonstrated that EFhd2 co‐localizes with pathological tau proteins in AD brains, confirming the co‐aggregation of EFhd2 and pathological tau. Furthermore, EFhd2's coiled‐coil domain mediated its self‐oligomerization in vitro and its association with tau proteins in JNPL3 mouse brain extracts. The results demonstrate that EFhd2 is a novel amyloid protein associated with pathological tau proteins in AD brain and that calcium binding may regulate the formation of EFhd2's amyloid structures. Hence, EFhd2 may play an important role in the pathobiology of tau‐mediated neurodegeneration.     

BAG-1M is up-regulated in hippocampus of Alzheimer's disease patients and associates with tau and APP proteins

The accumulation of tau and amyloid beta proteins is the major molecular pathology of Alzheimer's disease (AD). The mechanisms leading to the accumulation of these proteins are not completely clear. Hsc-70/Hsp-70, a chaperone protein, has been shown to bind both these proteins and regulate their degradation. We have previously shown that the co-chaperone protein BAG-1 can inhibit the degradation of tau by forming a complex with Hsc-70 and tau. In this current work, we show that there is an increase in the BAG-1M isoform in the hippocampus of AD patients. In addition, BAG-1 binds to both tau and amyloid precursor protein physically, and is found highly expressed in the same neurons that contain intracellular tau or amyloid in hippocampal sections from AD patients. Over-expression of BAG-1M in cell culture also induced an increase in both tau and amyloid precursor protein levels. In conclusion, we report a specific increase of BAG-1M in human AD patients, which is both physically and functionally associated to the two major molecular markers of AD.     

Altered tubulin assembly dynamics with N-homocysteinylated human 4R/1N tau in vitro

Tau isoforms promote neuronal integrity through binding and stabilization of microtubule proteins (MTP). It has been shown that hyperphosphorylation of tau contributes to Alzheimer’s disease (AD) pathology and related tauopathies. However, other pathogenic modifications of tau have not been well characterized. It is well accepted that elevated level of homocysteine (Hcy) is associated with neurodegenerative diseases such as AD. As a result of N-homocysteinylation of lysine residues, Hcy becomes a component of proteins, as a protein–homocystamide adduct, which affects protein structure and function. Here we demonstrate that N-homocysteinylation of human tau (4R/1N isoform) inhibits its function via impaired tau–tubulin specific binding and MTP assembly dynamics in vitro.     

Structural and functional implications of tau hyperphosphorylation: information from phosphorylation-mimicking mutated tau proteins

Abnormal tau-immunoreactive filaments are a hallmark of tauopathies, including Alzheimer's disease (AD). A higher phosphorylation ("hyperphosphorylation") state of tau protein may represent a critical event. To determine the potential role of tau hyperphosphorylation in these disorders, mutated tau proteins were produced where serine/threonine residues known to be highly phosphorylated in tau filaments isolated from AD patients were substituted for glutamate to simulate a paired helical filament (PHF)-like tau hyperphosphorylation. We demonstrate that, like hyperphosphorylation, glutamate substitutions induce compact structure elements and SDS-resistant conformational domains in tau protein. Hyperphosphorylation-mimicking glutamate-mutated tau proteins display a complete functional loss in its ability to promote microtubule nucleation which can partially be overcome by addition of the osmolyte trimethylamine N-oxide (TMAO), which is similar to phosphorylated tau. In addition, glutamate-mutated tau proteins fail to interact with the dominant brain protein phosphatase 2A isoform ABalphaC, and exhibit a reduced ability to assemble into filaments. Interestingly, wild-type tau and phosphorylation-mimicking tau similarly bind to microtubules when added alone, but the mutated tau is almost completely displaced from the microtubule surface by equimolar concentrations of wild-type tau. The data indicate that glutamate-mutated tau proteins provide a useful model for analyzing the functional consequences of tau hyperphosphorylation. They suggest that several mechanisms contribute to the abnormal tau accumulation observed during tauopathies, in particular a selective displacement of hyperphosphorylated tau from microtubules, a functional loss in promoting microtubule nucleation, and a failure to interact with phosphatases.     

Spreading of Alzheimer tau seeds is enhanced by aging and template matching with limited impact of amyloid-β

In Alzheimer''s disease (AD), deposition of pathological tau and amyloid-β (Aβ) drive synaptic loss and cognitive decline. The injection of misfolded tau aggregates extracted from human AD brains drives templated spreading of tau pathology within WT mouse brain. Here, we assessed the impact of Aβ copathology, of deleting loci known to modify AD risk (Ptk2b, Grn, and Tmem106b) and of pharmacological intervention with an Fyn kinase inhibitor on tau spreading after injection of AD tau extracts. The density and spreading of tau inclusions triggered by human tau seed were unaltered in the hippocampus and cortex of APPswe/PSEN1ΔE9 transgenic and App^NL-F/NL-F knock-in mice. In mice with human tau sequence replacing mouse tau, template matching enhanced neuritic tau burden. Human AD brain tau-enriched preparations contained aggregated Aβ, and the Aβ coinjection caused a redistribution of Aβ aggregates in mutant AD model mice. The injection-induced Aβ phenotype was spatially distinct from tau accumulation and could be ameliorated by depleting Aβ from tau extracts. These data suggest that Aβ and tau pathologies propagate by largely independent mechanisms after their initial formation. Altering the activity of the Fyn and Pyk2 (Ptk2b) kinases involved in Aβ-oligomer–induced signaling, or deleting expression of the progranulin and TMEM106B lysosomal proteins, did not alter the somatic tau inclusion burden or spreading. However, mouse aging had a prominent effect to increase the accumulation of neuritic tau after injection of human AD tau seeds into WT mice. These studies refine our knowledge of factors capable of modulating tau spreading.     

Two Monoclonal Antibodies Recognize Alzheimer''s Neurofibrillary Tangles, Neurofilament, and Microtubule-Associated Proteins

Two monoclonal antibodies that recognize Alzheimer's neurofibrillary tangles (ANTs), AD10 and AB18, have been characterized by immunoblotting against human and calf spinal cord neurofilament (NF) and calf brain microtubule preparations. Both antibodies bind to the 200-kilodalton (kd) (NF-H) and 160-kd (NF-M) but not to the 68-kd (NF-L) NF triplet proteins. They also bind to high-molecular-weight microtubule-associated proteins (MAPs) and tau. AD10 immunostains MAP2 and MAP1 families, whereas AB18 stains mainly MAP1 bands. Preincubation of intact filament preparation or nitrocellulose strips containing electroblotted NF proteins with Escherichia coli alkaline phosphatase completely blocks AD10 binding and partially blocks binding of AB18. These results suggest that the determinants recognized by these antibodies are phosphorylated. Immunoblotting of peptide fragments generated by limited proteolysis of NF proteins with alpha-chymotrypsin and Staphylococcus aureus V8 protease shows that the localization of the antigenic determinants to AD10 and AB18 in NF-H is approximately 100 and 60 kd, respectively, away from the carboxy terminal, a region previously shown to form the NF projection side arm. In NF-M, the antigenic determinants to both antibodies are located also in the projection side arm, in a 60-kd polypeptide adjacent to the alpha-helical filament core. The results show that ANTs contain at least two phosphorylated antigenic sites that are present in NF and MAPs, a finding suggesting that ANTs may be composed of proteins or their fragments with epitopes shared by cytoskeletal proteins.     

Pinning down phosphorylated tau and tauopathies

Neurofibrillary tangles (NFTs) are prominent neuronal lesions in a large subset of neurodegenerative diseases, including Alzheimer's disease (AD). NFTs are mainly composed of insoluble Tau that is hyperphosphorylated on many serine or threonine residues preceding proline (pSer/Thr-Pro). Tau hyperphosphorylation abolishes its biological function to bind microtubules and promotes microtubule assembly and precedes neurodegeneration. Not much is known about how tau is further regulated following phosphorylation. Notably, we have recently shown that phosphorylated Ser/Thr-Pro motifs exist in two distinct conformations. The conversion between two conformations in some proteins is catalyzed by the prolyl isomerase Pin1. Pin1 binds to tau phosphorylated specifically on the Thr231-Pro site and probably catalyzes cis/trans isomerization of pSer/Thr-Pro motif(s), thereby inducing conformational changes in tau. Such conformational changes can directly restore the ability of phosphorylated Tau to bind microtubules and promote microtubule assembly and/or facilitate tau dephosphorylation by its phosphatase PP2A, as PP2A activity is conformation-specific. Furthermore, Pin1 expression inversely correlates with the predicted neuronal vulnerability in normally aged brain and also with actual neurofibrillary degeneration in AD brain. Moreover, deletion of the gene encoding Pin1 in mice causes progressive age-dependent neuropathy characterized by motor and behavioral deficits, tau hyperphosphorylation, tau filament formation and neuronal degeneration. Distinct from all other mouse models where transgenic overexpression of specific proteins elicits tau-related pathologies, Pin1 is the first protein whose depletion causes age-dependent neurodegeneration and tau pathologies. Thus, Pin1 is pivotal in maintaining normal neuronal function and preventing age-dependent neurodegeneration. This could represent a promising interventive target to prevent neurodegenerative diseases.     

Interaction of tau isoforms with Alzheimer's disease abnormally hyperphosphorylated tau and in vitro phosphorylation into the disease-like protein

The microtubule-associated protein tau is a family of six isoforms that becomes abnormally hyperphosphorylated and accumulates in neurons undergoing neurodegeneration in the brains of patients with Alzheimer disease (AD). We investigated the isoform-specific interaction of normal tau with AD hyperphosphorylated tau (AD P-tau). We found that the binding of AD P-tau to normal human recombinant tau was tau4L > tau4S > tau4 and tau3L > tau3S > tau3, and that its binding to tau4L was greater than to tau3L. AD P-tau also inhibited the assembly of microtubules promoted by each tau isoform and caused disassembly when added to preassembled microtubules. This inhibition and depolymerization of microtubules by the AD P-tau corresponded directly to the degree of its interaction with the different tau isoforms. In vitro hyperphosphorylation of recombinant tau (P-tau) conferred AD P-tau-like characteristics. Like AD P-tau, P-tau interacted with and sequestered normal tau and inhibited microtubule assembly. These studies suggest that the AD P-tau interacts preferentially with the tau isoforms that have the amino-terminal inserts and four microtubule binding domain repeats and that hyperphosphorylation of tau appears to be sufficient to acquire AD P-tau characteristics. Thus, lack of amino-terminal inserts and extra microtubule binding domain repeat in fetal human brain might be protective from Alzheimer's neurofibrillary degeneration.     

Proteomic profiling of brain cortex tissues in a Tau transgenic mouse model of Alzheimer’s disease

Alzheimer’s disease (AD) involves regionalized neuronal death, synaptic loss, and an accumulation of intracellular neurofibrillary tangles and extracellular senile plaques. Although there have been numerous studies on tau proteins and AD in various stages of neurodegenerative disease pathology, the relationship between tau and AD is not yet fully understood. A transgenic mouse model expressing neuron-specific enolase (NSE)-controlled human wild-type tau (NSE-htau23), which displays some of the typical Alzheimer-associated pathological features, was used to analyze the brain proteome associated with tau tangle deposition. Two-dimensional electrophoresis was performed to compare the cortex proteins of transgenic mice (6- and 12-month-old) with those of control mice. Differentially expressed spots in different stages of AD were identified with ESI-Q-TOF (electrospray ionization quadruple time-of-flight) mass spectrometry and liquid chromatography/tandem mass spectrometry. Among the identified proteins, glutathione S-transferase P 1 (GSTP1) and carbonic anhydrase II (CAII) were down-regulated with the progression of AD, and secerin-1 (SCRN1) and V-type proton ATPase subunit E 1 (ATP6VE1) were up-regulated only in the early stages, and down-regulated in the later stages of AD. The proteins, which were further confirmed by RT-PCR at the mRNA level and with western blotting at the protein level, are expected to be good candidates as drug targets for AD. The study of up- and down-regulation of proteins during the progression of AD helps to explain the mechanisms associated with neuronal degeneration in AD.     

Characterization of a human and mouse tetrapyrrole-binding protein

The cDNA for p22HBP has been cloned from human and mouse, and the protein expressed, purified, and characterized. Both mouse and human proteins bind heme and porphyrins with micromolar K(d)s, are highly homologous, monomeric, and soluble, and have a cytoplasmic location. The proteins bind metalloporphyrins, free porphyrins, and N-methylprotoporphyrin with similar affinities, and mutations of a selected set of putative metal ligating residues did not have any significant effect on the measured K(d)s. That the presence or absence of metal in the porphyrin has no effect on the binding constants and the observation that the EPR signal for heme does not change upon binding to the protein strongly suggest that p22HBP is a generic tetrapyrrole-binding protein rather than a dedicated heme-binding protein. A role for p22HBP in cellular porphyrin metabolism is discussed.     

Characterization of two VQIXXK motifs for tau fibrillization in vitro

Tau proteins are building blocks of the filaments that form neurofibrillary tangles of Alzheimer's disease (AD) and related neurodegenerative tauopathies. It was recently reported that two VQIXXK motifs in the microtubule (MT) binding region, named PHF6 and PHF6*, are responsible for tau fibrillization. However, the exact role each of these motifs plays in this process has not been analyzed in detail. Using a recombinant human tau fragment containing only the four MT-binding repeats (K18), we show that deletion of either PHF6 or PHF6* affected tau assembly but only PHF6 is essential for filament formation, suggesting a critical role of this motif. To determine the amino acid residues within PHF6 that are required for tau fibrillization, a series of deletion and mutation constructs targeting this motif were generated. Deletion of VQI in either PHF6 or PHF6* lessened but did not eliminate K18 fibrillization. However, removal of the single K311 residue from PHF6 completely abrogated the fibril formation of K18. K311D mutation of K18 inhibited tau filament formation, while K311A and K311R mutations had no effect. These data imply that charge change at position 311 is important in tau fibril formation. A similar requirement of nonnegative charge at this position for fibrillization was observed with the full-length human tau isoform (T40), and data from these studies indicate that the formation of fibrils by T40K311D and T40K311P mutants is repressed at the nucleation phase. These findings provide important insights into the mechanisms of tau fibrillization and suggest targets for AD drug discovery to ameliorate neurodegeneration mediated by filamentous tau pathologies.     

Human sperm do not bind to rat zonae pellucidae despite the presence of four homologous glycoproteins

The specificity of sperm-egg recognition in mammals is mediated primarily by the zona pellucida surrounding ovulated eggs. Mouse sperm are quite promiscuous and bind to human eggs, but human spermatozoa will not bind to mouse eggs. The mouse zona pellucida contains three glycoproteins, ZP1, ZP2, and ZP3, which are conserved in rat and human. The recent observation that human zonae pellucidae contain a fourth protein raises the possibility that the presence of four zona proteins will support human sperm binding. Using mass spectrometry, four proteins that are similar in size and share 62-70% amino acid identity with human ZP1, ZP2, ZP3, and ZP4/ZPB were detected in rat zonae pellucidae. However, although mouse and rat spermatozoa bind to eggs from each rodent, human sperm bind to neither, and the presence of human follicular fluid did not alter the specificity of sperm binding. In addition, mutant mouse eggs lacking hybrid/complex N-glycans or deficient in Core 2 O-glycans were no more able to support human sperm binding than normal mouse eggs. These data suggest that the presence of four zona proteins are not sufficient to support human sperm binding to rodent eggs and that additional determinants must be responsible for taxon-specific fertilization among mammals.     

Widely divergent biochemical properties of the complete set of mouse DC-SIGN-related proteins

The mouse genome sequence has been examined to identify the complete set of proteins related to the human glycanbinding receptor, DC-SIGN. In addition to five SIGNR proteins previously described, a pseudogene, encoding a hypothetical SIGNR6, and a further two expressed proteins, SIGNR7 and SIGNR8, have been identified. The ligand-binding properties of these novel proteins and of the previously described mouse SIGNs have been systematically investigated in order to define the mouse proteins that most resemble human DC-SIGN and DC-SIGNR. Results from screening of a glycan array demonstrate that only mouse SIGNR3 shares with human DC-SIGN the ability to bind both high mannose and fucose-terminated glycans in this format and to mediate endocytosis. The finding that neither SIGNR1 nor SIGNR5 binds with high affinity to specific ligands in a large panel of mammalian glycans is consistent with the suggestion that these receptors bind surface polysaccharides on bacterial and fungal pathogens in a manner analogous to serum mannose-binding protein. The data also reveal that two of the mouse SIGNs have unusual binding specificities that have not been previously described for members of the C-type lectin family; the newly identified SIGNR7 binds preferentially to the 6-sulfo-sialyl Lewis(x) oligosaccharide, whereas SIGNR2 binds almost exclusively to glycans that bear terminal GlcNAc residues. The results presented demonstrate that the mouse homologs of DC-SIGN have a diverse set of ligand-binding and intracellular trafficking properties, some of which are distinct from the properties of any of the human receptors.     

Binding of copper (II) ion to an Alzheimer's tau peptide as revealed by MALDI-TOF MS, CD, and NMR

The tau protein plays an important role in some neurodegenerative diseases including Alzheimer's disease (AD). Neurofibrillary tangles (NFTs), a biological marker for AD, are aggregates of bundles of paired helical filaments (PHFs). In general, the alpha-sheet structure favors aberrant protein aggregates. However, some reports have shown that the alpha-helix structure is capable of triggering the formation of aberrant tau protein aggregates and PHFs have a high alpha-helix content. In addition, the third repeat fragment in the four-repeat microtubule-binding domain of the tau protein (residues 306-336: VQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQ, according to the longest tau protein) adopts a helical structure in trifluoroethanol (TFE) and may be a self-assembly model in the tau protein. In the human brain, there is a very small quantity of copper, which performs an important function. In our study, by means of matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS), circular dichroism (CD), and nuclear magnetic resonance (NMR) spectroscopy, the binding properties of copper (II) ion to the R3 peptide derived from the third repeat fragment (residues 318-335: VTSKCGSLGNIHHKPGGG) have been investigated. The results show that copper ions bind to the R3 peptide. CD spectra, ultraviolet (UV)-visible absorption spectra, and MALDI-TOF MS show pH dependence and stoichiometry of Cu2+ binding. Furthermore, CD spectra and NMR spectroscopy elucidate the copper binding sites located in the R3 peptide. Finally, CD spectra reveal that the R3 peptide adopts a mixture structure of random structures, alpha-helices, and beta-turns in aqueous solutions at physiological pH. At pH 7.5, the addition of 0.25 mol eq of Cu2+ induces the conformational change from the mixture mentioned above to a monomeric helical structure, and a beta-sheet structure forms in the presence of 1 mol eq of Cu2+. As alpha-helix and beta-sheet structures are responsible for the formation of PHFs, it is hypothesized that Cu2+ is an inducer of self-assembly of the R3 peptide and makes the R3 peptide form a structure like PHF. Hence, it is postulated that Cu2+ plays an important role in the aggregation of the R3 peptide and tau protein and that copper (II) binding may be another possible involvement in AD.     

Hyperphosphorylated tau in SY5Y cells: similarities and dissimilarities to abnormally hyperphosphorylated tau from Alzheimer disease brain.

Unlike normal tau, abnormally hyperphosphorylated tau (AD P-tau) from Alzheimer disease (AD) does not promote but instead inhibits microtubule assembly and disrupts already formed microtubules. Tau in the human neuroblastoma cell line SH-SY5Y is hyperphosphorylated at several of the same sites as AD P-tau, and accumulates in the cell body without any association to the cellular microtubule network. The aim of the present study was to elucidate why the SY5Y tau does not affect the viability of the cells. We found that, like AD P-tau, SY5Y tau because of hyperphosphorylation does not bind to microtubules and inhibits the tau-promoted assembly of microtubules. However, the tau/HMW MAP ratio is about 10 times less in SY5Y cells than in AD brain. These findings suggest that the hyperphosphorylated tau from SY5Y cells has similar biological characteristics as AD P-tau from AD brain, but is not lethal to the SY5Y cells because of its low tau/HMW MAP ratio.     

Alz 50, a monoclonal antibody to Alzheimer's disease antigen, cross-reacts with tau proteins from bovine and normal human brain

Microtubule-associated protein tau from bovine brain reacted on immunoblots and on enzyme-linked immunosorbent assay with a monoclonal antibody, Alz 50, which has previously been found to bind to an Alzheimer disease-specific antigen. The apparent affinity of binding of Alz 50 to tau was 2.1 X 10(-9) M on competitive enzyme-linked immunosorbent assay, and it was in the same range as for Tau-1 (0.5 X 10(-9) M), an antibody raised against purified bovine tau proteins. Immunoblotting of trypsin-digested tau revealed differences between Alz 50 and Tau-1 binding sites. The binding of both antibodies to tau was not affected by prior treatment with phosphatase, indicating that the cross-reactivity of Alz 50 with tau is due to the presence of phosphate-independent epitope. This epitope then differs from phosphate-dependent tau epitopes often shared with other cytoskeletal proteins. Alz 50 and Tau-1 binding sites were present in all isoelectric (pI 6-8) and molecular weight variants of tau. In contrast, phosphate-dependent epitopes recognized by another tau-reactive antibody (NP14) were found mostly in acidic tau variants. Similarly to tau proteins from bovine brain, tau-enriched preparations from normal human brain contained Alz 50 and Tau-1 reactive sites in all isoelectric (pI 6.5-8.5) and molecular weight variants. Our observation of Alz 50 cross-reactivity with tau suggests a relationship between tau and the novel protein identified recently in Alzheimer brains.     

Conformational shifts propagate from the oligomerization domain of p53 to its tetrameric DNA binding domain and restore DNA binding to select p53 mutants.

p53 is a conformationally flexible sequence-specific DNA binding protein mutated in many human tumors. To understand why the mutant p53 proteins associated with human tumors fail to bind DNA, we mapped the DNA binding domain of wild-type p53 and examined its regulation by changes in the protein conformation. Using site-directed mutagenesis, residues 90-286 of mouse p53 were shown to form the sequence-specific DNA binding domain. Two highly conserved regions within this domain, regions IV and V, were implicated in contacting DNA. Wild-type p53 bound DNA as a tetramer, each subunit recognizing five nucleotides of the 20 nucleotide-long DNA site. Conformational shifts of the oligomerization domain propagated to the tetrameric DNA binding domain, regulating DNA binding activity, but did not affect the subunit stoichiometry of wild-type p53 oligomers. Interestingly, conformational shifts could also be propagated within certain p53 mutants, rescuing DNA binding. One of these mutants was the mouse equivalent of human histidine 273, which is frequently associated with human tumors.     

Differential effects of an O-GlcNAcase inhibitor on tau phosphorylation

Abnormal hyperphosphorylation of microtubule-associated protein tau plays a crucial role in neurodegeneration in Alzheimer's disease (AD). The aggregation of hyperphosphorylated tau into neurofibrillary tangles is also a hallmark brain lesion of AD. Tau phosphorylation is regulated by tau kinases, tau phosphatases, and O-GlcNAcylation, a posttranslational modification of proteins on the serine or threonine residues with β-N-acetylglucosamine (GlcNAc). O-GlcNAcylation is dynamically regulated by O-GlcNAc transferase, the enzyme catalyzing the transfer of GlcNAc to proteins, and N-acetylglucosaminidase (OGA), the enzyme catalyzing the removal of GlcNAc from proteins. Thiamet-G is a recently synthesized potent OGA inhibitor, and initial studies suggest it can influence O-GlcNAc levels in the brain, allowing OGA inhibition to be a potential route to altering disease progression in AD. In this study, we injected thiamet-G into the lateral ventricle of mice to increase O-GlcNAcylation of proteins and investigated the resulting effects on site-specific tau phosphorylation. We found that acute thiamet-G treatment led to a decrease in tau phosphorylation at Thr181, Thr212, Ser214, Ser262/Ser356, Ser404 and Ser409, and an increase in tau phosphorylation at Ser199, Ser202, Ser396 and Ser422 in the mouse brain. Investigation of the major tau kinases showed that acute delivery of a high dose of thiamet-G into the brain also led to a marked activation of glycogen synthase kinase-3β (GSK-3β), possibly as a consequence of down-regulation of its upstream regulating kinase, AKT. However, the elevation of tau phosphorylation at the sites above was not observed and GSK-3β was not activated in cultured adult hippocampal progenitor cells or in PC12 cells after thiamet-G treatment. These results suggest that acute high-dose thiamet-G injection can not only directly antagonize tau phosphorylation, but also stimulate GSK-3β activity, with the downstream consequence being site-specific, bi-directional regulation of tau phosphorylation in the mammalian brain.     

The self-perpetuating tau truncation circle

Pathological truncations of human brain proteins represent the common feature of many neurodegenerative disorders including AD (Alzheimer's disease), Parkinson's disease and Huntington's disease. Protein truncations significantly change the structure and function of these proteins and thus can engender their pathological metamorphosis. We have shown previously that truncated forms of tau protein are contained in the core of the paired helical filaments that represent the main constituent of neurofibrillary pathology. Recently, we have identified truncated tau species of a different molecular signature. We have found that tau truncation is not produced by a random process, but rather by highly specific proteolytic cleavage and/or non-enzymatic fragmentation. In order to characterize the pathophysiology of AD-specific truncated tau species, we have used a transgenic rat model for AD expressing human truncated tau. Expression of the tau protein induces the formation of novel truncated tau species that originate from both transgenic human tau and endogenous rat tau proteins. Moreover, these truncated tau proteins are found exclusively in the misfolded fraction of tau, suggesting that they actively participate in the tau misfolding process. These findings corroborate further the idea that the appearance of truncated tau species starts a self-perpetuating cycle of further tau protein truncation leading to and accelerating tau misfolding and formation of neurofibrillary pathology.     

Copper (II) modulates in vitro aggregation of a tau peptide

Copper (II) has been implicated in the pathology of Alzheimer's disease (AD) for the impaired homeostatic mechanism found in the brains of AD patients. Here we studied the binding properties of Cu(II) with the first microtubule-binding repeat, encompassing residues 256-273 of the human tau441 sequence. Additionally, the effect of Cu(II) on the assembly of this repeat was also investigated. Our results indicate that Cu(II) can bind to this repeat with His(268) involved and has an inhibiting effect on the in vitro aggregation of this repeat. This work provides new insight into the role of Cu(II) in Alzheimer's disease.