Recombinant Tau Research Articles

Regulation of glycogen synthase kinase-3beta by products of lipid peroxidation in human neuroblastoma cells.

The potential role of 4-hydroxynonenal (HNE), a major product of membrane lipid peroxidation, in regulating glycogen synthase kinase-3beta (GSK3beta) activity was examined in human neuroblastoma IMR-32 cells. The inhibition of GSK3beta activity by HNE was observed by in vitro kinase assays with two substrates, the synthetic glycogen synthase peptide-2 and the human recombinant tau. GSK3beta activity is regulated by Ser9 (inhibitory) and Tyr216 (stimulatory) phosphorylation. By using specific activity-dependent phospho-antibodies, immunoblot analysis revealed that HNE induces an increase in phosphorylation of GSK3beta in Ser9, enhancing basal phosphatidylinositol 3-kinase (PI3K)/AKT and extracellular signal-regulated kinase 2 (ERK2) signalling pathways. Ser9-GSK3beta phosphorylation induced by HNE was abolished by treatment with LY294002 or U0126, two inhibitors of PI3K/AKT and ERK pathways, respectively. These experiments provide evidence for a crucial role of the PI3K/AKT and ERK2 pathways as intracellular targets of HNE that mediate the inhibition of GSK3beta activity in regulating cellular response to HNE in viable cells under conditions in which membrane lipid peroxidation occurs. These data support a key role for GSK3beta as a mediator of the signalling pathways activated by oxidative stress, and therefore it may be included among the redox-sensitive enzymes.

Surface-decoration of Microtubules by Human Tau

Tau is a neuronal, microtubule-associated protein that stabilizes microtubules and promotes neurite outgrowth. Tau is largely unfolded in solution and presumably forms mostly random coil. Because of its hydrophilic nature and flexible structure, tau complexed to microtubules is largely invisible by standard electron microscopy methods. We applied a combination of high-resolution metal-shadowing and cryo-electron microscopy to study the interactions between tau and microtubules. We used recombinant tau variants with different domain compositions, (1) full length tau, (2) the repeat domain that mediates microtubule binding (K19), and (3) two GFP-tau fusion proteins that contain a globular marker (GFP) attached to full-length tau at either end. All of these constructs bind exclusively to the outside of microtubules. Most of the tau-related mass appears randomly distributed, creating a “halo” of low-density mass spread across the microtubule surface. Only a small fraction of tau creates a periodic signal at an 8 nm interval, centered on α-tubulin subunits. Our data suggest that tau retains most of its disordered structure even when bound to the microtubule surface. Hence, it binds along, as well as across protofilaments. Nevertheless, even minute concentrations of tau have a strong stabilizing effect and effectively scavenge unpolymerized tubulin.

Pseudophosphorylation of tau protein alters its ability for self-aggregation.

Filamentous tau protein deposits are a pathological hallmark of a group of neurodegenerative disorders (tauopathies). Tau protein in these aggregates is highly phosphorylated at different phosphorylation sites. Although tau filaments can be formed by heparin-induced aggregation of unphosphorylated recombinant tau, it is not known how tau phosphorylation modulates aggregation behaviour. Analysis of the effect of tau phosphorylation at defined single or multiple sites is hampered by the low specificity of protein kinases and the highly dynamic turnover of phosphorylation in vivo. To overcome this problem we employed site-directed mutagenesis to convert serine and threonine to aspartic acid or glutamic acid, which introduce a negative charge and conformational change that mimic phosphorylation. We tested 14 different mutated tau proteins for their propensity for self-aggregation and formation of tau filaments. Tau aggregation was monitored with thioflavin S fluorescence in the presence of different inducers such as heparin, Al3+, Fe2+ and Fe3+. We found that mutations in the N-terminal portion up to amino acid 208 mainly suppress tau aggregation, whereas mutations in the C-terminal region mainly lead to an enhanced aggregation. Mutations in the middle portion of tau showed a mixed picture of suppression and enhancement of aggregation. A single amino acid change Ser422Glu has aggregation-favouring properties with all four inducers.

Tau paired helical filaments from Alzheimer's disease brain and assembled in vitro are based on beta-structure in the core domain.

Tau protein, a neuronal microtubule-associated protein, forms insoluble fibers ("paired helical filaments") in Alzheimer's disease and other tauopathies. Conflicting views on the structure of the fibers have been proposed recently, ranging from mainly alpha-helical structure to mainly beta-sheet, or a mixture of mostly random coil and beta-sheet. We have addressed this issue by studying tau fibers immunopurified from Alzheimer brain tissue by a conformation-specific antibody and comparing them with fibers reassembled from recombinant tau or tau constructs in vitro, using a combination of electron microscopy and spectroscopic methods. Brain-derived fibers and reassembled fibers both exhibit a typical twisted appearance when examined by electron microscopy. The soluble tau protein is a natively unfolded protein dominated by random coil structure, whereas Alzheimer PHFs and reassembled fibers show a shift toward an increase in the level of beta-structure. The results support a model in which the repeat domain of tau (which lies within the core of PHFs) adopts an increasing level of beta-structure during aggregation, whereas the N- and C-terminal domains projecting away from the PHF core are mostly random coil.

Dephosphorylation of microtubule-associated protein tau by protein phosphatase 5.

Protein phosphatase 5 (PP5) is a 58-kDa novel phosphoseryl/phosphothreonyl protein phosphatase. It is ubiquitously expressed in all mammalian tissues examined, with a high level in the brain, but little is known about its physiological substrates. We found that this phosphatase dephosphorylated recombinant tau phosphorylated with cAMP-dependent protein kinase and glycogen synthase kinase-3beta, as well as abnormally hyperphosphorylated tau isolated from brains of patients with Alzheimer's disease. The specific activity of PP5 toward tau was comparable to those reported with other protein substrates examined to date. The PP5 activity toward tau was stimulated by arachidonic acid by 30- to 45-fold. Immunostaining demonstrated that PP5 was primarily cytoplasmic in PC12 cells and in neurons of postmortem human brain tissue. A small pool of PP5 associated with microtubules. Expression of active PP5 in PC12 cells resulted in reduced phosphorylation of tau, suggesting that PP5 can also dephosphorylate tau in cells. These results suggest that PP5 plays a role in the dephosphorylation of tau and might be involved in the molecular pathogenesis of Alzheimer's disease.

Phosphorylation of tau protein to sites found in Alzheimer's disease brain is catalyzed by Ca 2+/calmodulin-dependent protein kinase II as demonstrated tandem mass spectrometry

Neuronal Ca 2+/calmodulin-dependent protein kinase II (CaMKII) is one of the most abundant protein kinases in the brain, and phosphorylates a broad range of substrate proteins. The phosphorylation of microtubule tau by CaMKII was investigated using tandem mass spectrometry (MS/MS). Recombinant human tau was phosphorylated at Thr212, Ser214, Ser262, and Ser356 by CaMKII. The phosphorylation of these sites is found in paired helical filament (PHF)-tau. In addition to these sites, Ser131 and Thr135 were phosphorylated by CaMKII. Phosphorylation at Ser131, Thr135, Thr212 and Ser214 by CaMKII has not been reported previously. Thr212 and Ser214 are in the consensus phosphorylation sequence of CaMKII (RXXS/T), and non-fetal-type phosphorylation sites of tau. Non-fetal-type phosphorylation may produce PHF-tau. These results suggested that CaMKII is involved in the phosphorylation of tau in Alzheimer's disease brain.

Anionic Micelles and Vesicles Induce Tau Fibrillization in Vitro

Alzheimer's disease is defined in part by the intraneuronal accumulation of filaments comprised of the microtubule-associated protein tau. In vitro, fibrillization of recombinant tau can be induced by treatment with various agents, including phosphotransferases, polyanionic compounds, and fatty acids. Here we characterize the structural features required for the fatty acid class of tau fibrillization inducer using recombinant full-length tau protein, arachidonic acid, and a series of straight chain anionic, cationic, and nonionic detergents. Induction of measurable tau fibrillization required an alkyl chain length of at least 12 carbons and a negative charge consisting of carboxylate, sulfonate, or sulfate moieties. All detergents and fatty acids were micellar at active concentrations, due to a profound, taudependent depression of their critical micelle concentrations. Anionic surfaces larger than detergent micelles, such as those supplied by phosphatidylserine vesicles, also induced tau fibrillization with resultant filaments originating from their surface. These data suggest that anionic surfaces presented as micelles or vesicles can serve to nucleate tau fibrillization, that this mechanism underlies the activity of fatty acid inducers, and that anionic membranes may serve this function in vivo.

Proteasome inhibition by paired helical filament‐tau in brains of patients with Alzheimer's disease

Alzheimer's disease (AD) is characterized neuropathologically by intracellular neurofibrillary tangles (NFTs) formed of tau-based paired helical filaments (PHFs) and extracellular beta-amyloid plaques. The degree of Alzheimer dementia correlates with the severity of PHFs and NFTs. As an intraneuronal accumulation of oxidatively damaged proteins has been found in the brains of patients with AD, a dysfunction of the proteasomal system, which degrades damaged proteins, has been assumed to cause protein aggregation and therefore neurodegeneration in AD. In this study, we revealed that such proteasome dysfunction in AD brain results from the inhibitory binding of PHF-tau to proteasomes. We analysed the proteasome activity in brains from patients with AD and age-matched controls, and observed a significant decrease to 56% of the control level in the straight gyrus of patients with AD. This loss of activity was not associated with a decrease in the proteasome protein. PHF-tau co-precipitated during proteasome immunoprecipitation and proteasome subunits could be co-isolated during isolation of PHFs from AD brain. Furthermore, the proteasome activity in human brains strongly correlated with the amount of co-precipitated PHF-tau during immunoprecipitation of proteasome. Incubation of isolated proteasomes with PHF-tau isolated from AD brain, and with PHFs after in vitro assembly from human recombinant tau protein, resulted in a distinct inhibition of proteasome activity by PHF-tau. As this inhibition of proteasome activity was sufficient to induce neuronal degeneration and death, we suggest that PHF-tau is able directly to induce neuronal damage in the AD brain.

Glycogen Synthase Kinase 3β Phosphorylates Tau at Both Primed and Unprimed Sites: DIFFERENTIAL IMPACT ON MICROTUBULE BINDING

Glycogen synthase kinase 3beta (GSK3beta) phosphorylates substrates, including the microtubule-associated protein tau, at both primed and unprimed epitopes. GSK3beta phosphorylation of tau negatively regulates tau-microtubule interactions; however the differential effects of phosphorylation at primed and unprimed epitopes on tau is unknown. To examine the phosphorylation of tau at primed and unprimed epitopes and how this impacts tau function, the R96A mutant of GSK3beta was used, a mutation that prevents phosphorylation of substrates at primed sites. Both GSK3beta and GSK3beta-R96A phosphorylated tau efficiently in situ. However, expression of GSK3beta-R96A resulted in significantly less phosphorylation of tau at primed sites compared with GSK3beta. Conversely, GSK3beta-R96A phosphorylated unprimed tau sites to a significantly greater extent than GSK3beta. Prephosphorylating tau with cdk5/p25 impaired the ability of GSK3beta-R96A to phosphorylate tau, whereas GSK3beta-R96A phosphorylated recombinant tau to a significantly greater extent than GSK3beta. Moreover, the amount of tau associated with microtubules was reduced by overexpression of GSK3beta but only when tau was phosphorylated at primed sites, as phosphorylation of tau by GSK3beta-R96A did not negatively regulate the association of tau with microtubules. These results demonstrate that GSK3beta-mediated phosphorylation of tau at primed sites plays a more significant role in regulating the interaction of tau with microtubules than phosphorylation at unprimed epitopes.

Involvement of aberrant glycosylation in phosphorylation of tau by cdk5 and GSK-3β

Microtubule-associated protein tau is abnormally hyperphosphorylated, glycosylated, and aggregated in affected neurons in the brains of individuals with Alzheimer’s disease (AD). We recently found that the glycosylation might precede hyperphosphorylation of tau in AD. In this study, we investigated the effect of glycosylation on phosphorylation of tau catalyzed by cyclin-dependent kinase 5 (cdk5) and glycogen synthase kinase-3β (GSK-3β). The phosphorylation of the longest isoform of recombinant human brain tau, tau 441, at various sites was detected by Western blots and by radioimmuno-dot-blot assay with phosphorylation-dependent and site-specific tau antibodies. We found that cdk5 phosphorylated tau 441 at Thr-181, Ser-199, Ser-202, Thr-205, Thr-212, Ser-214, Thr-217, Thr-231, Ser-235, Ser-396, and Ser-404, but not at Ser-262, Ser-400, Thr-403, Ser-409, Ser-413, or Ser-422. GSK-3β phosphorylated all the cdk5-catalyzed sites above except Ser-235. Deglycosylation by glycosidases depressed the subsequent phosphorylation of AD-tau (i) with cdk5 at Thr-181, Ser-199, Ser-202, Thr-205, and Ser-404, but not at Thr-212; and (ii) with GSK-3β at Thr-181, Ser-202, Thr-205, Ser-217, and Ser-404, but not at Ser-199, Thr-212, Thr-231, or Ser-396. These data suggest that aberrant glycosylation of tau in AD might be involved in neurofibrillary degeneration by promoting abnormal hyperphosphorylation by cdk5 and GSK-3β.

Evidence that absence of endometrial gland secretions in uterine gland knockout ewes compromises conceptus survival and elongation

Endometrial glands are necessary for conceptus implantation and growth. In the ovine uterine gland knockout (UGKO) model, blastocysts hatch normally but fail to survive or elongate. This peri-implantation defect in UGKO ewes may be due to the absence of endometrial glands or, alternatively, to the lack of certain epithelial adhesion molecules or the inability of the endometrium to respond to signals from the conceptus. Two studies were performed to examine these hypotheses. In study one, normal (n = 8) and UGKO (n = 12) ewes were mated at oestrus (day 0) with intact rams and their uteri were flushed 14 days after oestrus. Normal ewes (n = 4) were also flushed on 14 days after oestrus. Uterine flushes from bred normal ewes contained filamentous conceptuses (n = 7 of 8), whereas those from UGKO ewes contained no conceptus (n = 5 of 12), a growth-retarded, tubular conceptus (n = 6 of 12), or a fragmented, filamentous conceptus (n = 1 of 12). In all groups, expression of mucin 1 and integrin alpha(v), alpha(5), beta(3) and beta(5) was localized at the apical surface of the endometrial luminal epithelium with no detectable differences between normal and UGKO ewes. Uterine flushes from pregnant ewes, but not cyclic or UGKO ewes, contained abundant immunoreactive interferon tau and the cell adhesion proteins, osteopontin and glycosylation-dependent cell adhesion molecule one. In study two, UGKO ewes were fitted with uterine catheters 5 days after oestrus, infused with recombinant ovine interferon tau or control proteins from 11 to 15 days after oestrus, and underwent hysterectomy 16 days after oestrus. Expression of several interferon tau-stimulated genes (ISG17, STAT1, STAT2 and IRF-1) was increased in the endometrium from interferon tau-infused UGKO ewes. These results support the hypothesis that the defects in conceptus elongation and survival in UGKO ewes are due to the absence of endometrial glands and their secretions rather than to alterations in expression of anti-adhesive or adhesive molecules on the endometrial luminal epithelium or to the responsiveness of the endometrium to the conceptus pregnancy recognition signal.

Evidence that absence of endometrial gland secretions in uterine gland knockout ewes compromises conceptus survival and elongation

Endometrial glands are necessary for conceptus implantation and growth. In the ovine uterine gland knockout (UGKO) model, blastocysts hatch normally but fail to survive or elongate. This peri-implantation defect in UGKO ewes may be due to the absence of endometrial glands or, alternatively, to the lack of certain epithelial adhesion molecules or the inability of the endometrium to respond to signals from the conceptus. Two studies were performed to examine these hypotheses. In study one, normal (n = 8) and UGKO (n = 12) ewes were mated at oestrus (day 0) with intact rams and their uteri were flushed 14 days after oestrus. Normal ewes (n = 4) were also flushed on 14 days after oestrus. Uterine flushes from bred normal ewes contained filamentous conceptuses (n = 7 of 8), whereas those from UGKO ewes contained no conceptus (n = 5 of 12), a growth-retarded, tubular conceptus (n = 6 of 12), or a fragmented, filamentous conceptus (n = 1 of 12). In all groups, expression of mucin 1 and integrin alpha(v), alpha(5), beta(3) and beta(5) was localized at the apical surface of the endometrial luminal epithelium with no detectable differences between normal and UGKO ewes. Uterine flushes from pregnant ewes, but not cyclic or UGKO ewes, contained abundant immunoreactive interferon tau and the cell adhesion proteins, osteopontin and glycosylation-dependent cell adhesion molecule one. In study two, UGKO ewes were fitted with uterine catheters 5 days after oestrus, infused with recombinant ovine interferon tau or control proteins from 11 to 15 days after oestrus, and underwent hysterectomy 16 days after oestrus. Expression of several interferon tau-stimulated genes (ISG17, STAT1, STAT2 and IRF-1) was increased in the endometrium from interferon tau-infused UGKO ewes. These results support the hypothesis that the defects in conceptus elongation and survival in UGKO ewes are due to the absence of endometrial glands and their secretions rather than to alterations in expression of anti-adhesive or adhesive molecules on the endometrial luminal epithelium or to the responsiveness of the endometrium to the conceptus pregnancy recognition signal.

Direct interaction of soluble human recombinant tau protein with Aβ 1–42 results in tau aggregation and hyperphosphorylation by tau protein kinase II

We report here that aggregated β-amyloid (Aβ) 1–42 promotes tau aggregation in vitro in a dose-dependent manner. When Aβ-mediated aggregated tau was used as a substrate for tau protein kinase II (TPK II), an 8-fold increase in the rate of TPK II-mediated tau phosphorylation was observed. The extent of TPK II-dependent tau phosphorylation increased as a function of time and Aβ 1–42 concentration, and hyperphosphorylated tau was found to be decorated with an Alzheimer’s disease-related phosphoepitope (P-Thr-231). In HEK 293 cells co-expressing CT-100 amyloid precursor protein and tau, the release of Aβ 1–42 from these cells was impaired. Taken together, these in vitro results suggest that Aβ 1–42 promotes both tau aggregation and hyperphosphorylation.

Effects on splicing and protein function of three mutations in codon N296 of tau in vitro

Three Mutations were recently reported in the same codon (N296) in exon 10 of the tau gene. Two of these mutations, N296N and N296H, lead to a clinical syndrome similar to autosomal dominant fronto-temporal dementia with Parkinsonism linked to chromosome 17. In contrast the third mutation, delN296, gives rise to atypical progressive supranuclear palsy in individuals homozygous for the mutation, but in heterozygous individuals this mutation is incompletely penetrant and associated with a phenotype similar to idiopathic Parkinson's disease. Functional assays were employed to determine the effects of these mutations on alternative splicing of exon 10, on microtubule assembly and self-aggregation of recombinant tau protein. We demonstrate that these mutations exhibit a spectrum of potentially pathogenic changes in tau function, and provide insight into the possible cause of the incompletely penetrant phenotype of the delN296 mutation.

Testosterone prevents the heat shock-induced overactivation of glycogen synthase kinase-3 beta but not of cyclin-dependent kinase 5 and c-Jun NH2-terminal kinase and concomitantly abolishes hyperphosphorylation of tau: implications for Alzheimer's disease.

We have shown previously that glycogen synthase kinase-3 beta (GSK-3 beta), cyclin-dependent kinase 5, and c-Jun NH(2)-terminal kinase become overactivated and hyperphosphorylate tau in heat-shocked female rats. This hyperphosphorylation of tau is estrogen-independent, prevented by androgens, and similar to Alzheimer's disease. In this study, ovariectomized (OVX) Sprague-Dawley rats (n = 75) received daily injections of 10 microg of 17 beta-estradiol benzoate (EB), or 250 microg of testosterone propionate (TP), or both EB and TP, or sesame oil (SO) vehicle for 4-6 weeks. In kinase assays of forebrain homogenates, overactivation of GSK-3 beta at 0-6 h after heat shock toward human recombinant tau, bovine tau, and phosphoglycogen synthase peptide 2 was prevented in OVX + TP and OVX + (EB + TP) but not in sham-OVX + SO, OVX + SO, and OVX + EB. Abs against inactive (pSer(9)) and activity-enhanced (pTyr(216)) GSK-3 beta showed marked increase of pSer(9)- and decrease of pTyr(216)-GSK-3 beta in both OVX + TP and OVX + (EB + TP) but not in sham-OVX + SO, OVX + SO, and OVX + EB. EB enhanced the overactivation of cyclin-dependent kinase 5. The activity of c-Jun NH(2)-terminal kinase was gonadal hormone-independent. The serum concentrations of testosterone and 17 beta-estradiol were 2.53 ng/ml and 201 pg/ml in OVX + TP and OVX + EB, respectively. These findings demonstrate that testosterone prevents the hyperphosphorylation of tau by inhibiting the heat shock-induced overactivation of GSK-3 beta and suggest that androgens given to aging men or, in combination with estrogens, to postmenopausal women could prevent or delay Alzheimer's disease.

A Raman optical activity study of rheomorphism in caseins, synucleins and tau. New insight into the structure and behaviour of natively unfolded proteins.

The casein milk proteins and the brain proteins alpha-synuclein and tau have been described as natively unfolded with random coil structures, which, in the case of alpha-synuclein and tau, have a propensity to form the fibrils found in a number of neurodegenerative diseases. New insight into the structures of these proteins has been provided by a Raman optical activity study, supplemented with differential scanning calorimetry, of bovine beta- and kappa-casein, recombinant human alpha-, beta- and gamma-synuclein, together with the A30P and A53T mutants of alpha-synuclein associated with familial cases of Parkinson's disease, and recombinant human tau 46 together with the tau 46 P301L mutant associated with inherited frontotemporal dementia. The Raman optical activity spectra of all these proteins are very similar, being dominated by a strong positive band centred at approximately 1318 cm(-1) that may be due to the poly(l-proline) II (PPII) helical conformation. There are no Raman optical activity bands characteristic of extended secondary structure, although some unassociated beta strand may be present. Differential scanning calorimetry revealed no thermal transitions for these proteins in the range 15-110 degrees C, suggesting that the structures are loose and noncooperative. As it is extended, flexible, lacks intrachain hydrogen bonds and is hydrated in aqueous solution, PPII helix may impart a rheomorphic (flowing shape) character to the structure of these proteins that could be essential for their native function but which may, in the case of alpha-synuclein and tau, result in a propensity for pathological fibril formation due to particular residue properties.

Interaction of Tau Isoforms with Alzheimer's Disease Abnormally Hyperphosphorylated Tau and in VitroPhosphorylation 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.

Pick's disease associated with the novel Tau gene mutation K369I.

Exonic and intronic mutations in Tau cause neurodegenerative syndromes characterized by frontotemporal dementia and filamentous tau protein deposits. We describe a K369I missense mutation in exon 12 of Tau in a patient with a pathology typical of sporadic Pick's disease. The proband presented with severe personality changes, followed by loss of cognitive function. Detailed postmortem examination of the brain showed atrophy, which was most pronounced in the temporal lobes; and numerous tau-immunoreactive Pick bodies and Pick cells in the neocortex and the hippocampal formation, as well as in subcortical brain regions. Their appearance and staining characteristics were indistinguishable from those of sporadic Pick's disease. However, immunoblot analysis of sarkosyl-insoluble tau showed three major bands of 60, 64, and 68 kDa, consistent with the presence of 3- and 4-repeat tau isoforms, as in Alzheimer's disease. Isolated tau filaments were irregularly twisted ribbons, with a small number of Alzheimer-type paired helical filaments. In the presence of heparin, tau proteins with the K369I mutation formed short, slender filaments. Biochemically, recombinant tau proteins with the K369I mutation showed reduced ability to promote microtubule assembly, suggesting that this may be the primary effect of the mutation by providing a pool of aberrant tau for filament assembly. Taken together, results indicate that the K369I mutation in Tau can cause a dementing disease with a neuropathology like that of Pick's disease.

Expression of the antiviral protein Mx in peripheral blood mononuclear cells of pregnant and bred, non-pregnant ewes.

Interferon-tau (IFN tau) acts locally on the endometrium to suppress estrogen and oxytocin receptor expression and block luteolysis in ruminants. Systemic administration of conceptus homogenates or recombinant ovine IFN tau does not block luteolysis or enhance pregnancy rates in sheep or cattle, respectively. However, IFN tau up-regulates expression of the antiviral protein Mx throughout the entire uterine wall during early pregnancy. These studies determined if conceptus-derived IFN tau also up-regulates Mx expression in components of the circulating immune system that migrate through the endometrial wall. In experiment one, peripheral blood mononuclear cells (PBMC) were isolated from ewes at D26 post-artificial insemination (AI) and Mx mRNA levels examined by Northern and slot-blot hybridization. Pregnancy resulted in a two-fold increase in Mx mRNA levels compared to bred, non-pregnant ewes at D26. In experiment two, PBMC were isolated from ewes at AI, and every three days from D9 to D30. Results showed a four-fold increase in Mx mRNA levels in PBMC from pregnant versus bred, non-pregnant ewes at D15. Increased Mx mRNA, which remained elevated through D30, was accompanied by increased levels of Mx protein. These results show that pregnancy recognition signaling rapidly induces Mx gene expression in PBMC, and are the first to suggest that IFN tau activates gene expression in components of the circulating immune system.

Role of Cysteine-291 and Cysteine-322 in the Polymerization of Human Tau into Alzheimer-like Filaments

Filamentous tau pathology is central to a large number of dementing disorders, including Alzheimer's disease in which polymerized tau is hyperphosphorylated. Previous studies on heparin-dependent tau polymerization, using recombinant tau isoforms lacking Cys-291, suggest that tau dimerization via Cys-322 is critical for initiation of assembly of soluble tau into filaments. We report heparin-dependent in vitro polymerization of human recombinant tau (1–383 isoform), containing both Cys-291 and Cys-322, into paired helical filaments as characterized by electron microscopy. Tau polymerization, under physiological tau concentrations in the presence of dithiothreitol (DTT), was followed by a Thioflavine S fluorescence assay. To understand the molecular basis for heparin-induced tau polymerization, we expressed and purified C291A, C322A, and C291A/C322A tau mutants. The DTT requirement for tau polymerization was abolished using either the C291A or C322A tau mutant and polymerization was not observed with the C291A/C322A tau double mutant. Analysis by sodium dodecyl sulfate gel electrophoresis showed that, unlike wild type tau, a significant amount of the C291A mutant and the C322A mutant is present as a disulfide bonded dimer. Taken together these results suggest that, in isoforms containing both Cys-291 and Cys-322, a dimeric tau with an intermolecular disulfide bond through either Cys-291 or Cys-322 is presumably acting as a seed for initiation of tau polymerization.