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.     

Neighbored phosphorylation sites as PHF-tau specific markers in Alzheimer's disease

Neurofibrillary tangles, which represent a major pathological hallmark in Alzheimer's disease (AD), are deposits of the hyperphosphorylated microtubule-associated tau protein (PHF-tau). However, a link between the phosphorylation pattern and the cause or the progress of AD is still missing. The work reported here focused on PHF-tau specific local phosphorylation patterns at Thr212/Ser214 and Thr231/Ser235 using monoclonal antibodies (mAb) generated against correspondingly modified peptides. The binding motifs of the obtained six mAbs were characterized with non-, mono-, and double-phosphorylated peptides as well as terminally shortened sequences. Five mAbs stained neurofibrillary tangles, neuritic plaques, and neuropil threads from autoptic brains of AD cases. Four mAbs recognized PHF-tau without significant cross-reactivity towards normal human tau, bovine tau, and dephosphorylated PHF-tau in ELISA and Western blot analysis. Thus, double phosphorylation is sufficient to distinguish PHF-tau from all other tau versions and there is no need to postulate any PHF-tau specific conformation for this region.     

Tau Released from Paired Helical Filaments with Formic Acid or Guanidine Is Susceptible to Calpain-Mediated Proteolysis

Abstract: Paired helical filaments (PHFs), a characteristic neuropathologic finding in Alzheimer's disease brain, are abnormal fibrillary forms of hyperphosphorylated tau (PHF-tau), which have been shown to be highly resistant to calpain digestion. Either excessive phosphorylation or fibrillary arrangement of tau proteins in PHFs may play a role in proteolytic resistance by limiting access to calpain recognition/digestion sites. To determine the contribution of the fibrillary conformation, isolated PHFs were subjected to treatment with either formic acid or guanidine. Both procedures effectively abolished the fibrillary structure of PHF but preserved PHF-tau immunoreactivity using a panel of antibodies that recognize nonphosphorylated and phosphorylated epitopes. These treatments also significantly increased the sensitivity of PHF-tau polypeptides to calpain proteolysis as shown by significant decreases in the half-life ( t _1/2) from the infinite with native PHF to 44 min and 4.4 min in formic acid- or guanidine-treated samples, respectively. In contrast, the sensitivity of normal fetal tau (3.4 min) was either decreased (5.9 min) or unaffected (3.6 min) by similar treatment. Our results indicate that after guanidine treatment, the sensitivity of PHF to calpain resembles that of fetal tau. These results strongly suggest that the fibrillary structure of PHF-tau, rather than hyperphosphorylation, is the major factor responsible for the resistance of abnormal filaments to calpain-mediated proteolysis.     

Alzheimer's disease-like phosphorylation of the microtubule-associated protein tau by glycogen synthase kinase-3 in transfected mammalian cells

Background: Paired helical filaments (PHFs) are a characteristic pathological feature of Alzheimer's disease; their principal component is the microtubule-associated protein tau. The tau in PHFs (PHF-tau) is hyperphosphorylated, but the cellular mechanisms responsible for this hyperphosphorylation have yet to be elucidated. A number of kinases, including mitogen-activated protein (MAP) kinase, glycogen synthase kinase (GSK)-3α, GSK-3β and cyclin-dependent kinase-5, phosphorylate recombinant tau in vitro so that it resembles PHF-tau as judged by its reactivity with a panel of antibodies capable of discriminating between normal tau and PHF-tau, and by a reduced electrophoretic mobility that is characteristic of PHF-tau. To determine whether MAP kinase, GSK-3α and GSK-3β can also induce Alzheimer's disease-like phosphorylation of tau in mammalian cells, we studied the phosphorylation status of tau in primary neuronal cultures and transfected COS cells following changes in the activities of MAP kinase and GSK-3.Results Activating MAP kinase in cultures of primary neurons or transfected COS cells expressing tau isoforms did not increase the level of phosphorylation for any PHF-tau epitope investigated. But elevating GSK-3 activity in the COS cells by co-transfection with GSK-3α or GSK-3β decreased the electrophoretic mobility of tau so that it resembled that of PHF-tau, and induced reactivity with eight PHF-tau-selective monoclonal antibodies.Conclusion Our data indicate that GSK-3α and/or GSK-3β, but not MAP kinase, are good candidates for generating PHF-type phosphorylation of tau in Alzheimer's disease. The involvement of other kinases in the generation of PHFs cannot, however, be eliminated. Our results suggest that aberrant regulation of GSK-3 may be a pathogenic mechanism in Alzheimer's disease.     

Epitope mapping of mAbs AT8 and Tau5 directed against hyperphosphorylated regions of the human tau protein

Post-mortem diagnosis of Alzheimer's disease relies on high numbers of senile plaques and neurofibrillary tangles (NFTs) stained in distinct brain areas. NFTs mostly consist of hyperphosphorylated versions of the microtubule attached tau protein (PHF-tau) with more than 30 serine and threonine phosphorylation sites identified so far. Characterization of hyperphosphorylated tau regions and the hope to develop robust assays for early AD diagnosis relies mostly on phosphorylation-dependent monoclonal antibodies (mAbs) recognizing only disease-specific phosphorylation patterns. Here, we report that anti-PHF-tau mAb AT8 recognizes an epitope doubly phosphorylated at serine 202 and threonine 205, which was not influenced by a third phosphate group at serine 199. But mAb AT8 was cross-reactive to two doubly phosphorylated motifs containing either serines 199 and 202 or serines 205 and 208 of the human tau sequence. The epitope of anti-tau mAb Tau5 was mapped to the human tau sequence 218-225, which is not phosphorylated in vivo.     

Structural stability of paired helical filaments requires microtubule-binding domains of tau: a model for self-association.

Highly purified and SDS-soluble paired helical filaments (PHFs) were immunogold labeled and immunoblotted with antibodies to tau: Tau 14 (N-terminal half), AH-1 (microtubule-binding domain), and Tau 46 (C-terminal end). The main component of PHFs was modified tau of 68, 64, and 60 kd, also called A68 or PHF-tau. Trypsin digestion reduced the maximum width of PHFs by 10%-20%, increased aggregation of filaments, and abolished the binding of Tau 14, but had no effect on the binding of AH-1. The smallest tau-reactive tryptic fragments were 13 and 7-8 kd, positive with AH-1, and negative with Tau 46. Our results and the model of Crowther and Wischik suggest that by self-association and anti-parallel arrangement of the microtubule-binding domains, PHF-tau forms the backbone of PHFs.     

Alzheimer-specific epitopes of tau represent lipid peroxidation-induced conformations

Several recent studies support a link between tau protein phosphorylation and adduction of tau by reactive carbonyls. Indeed, the phosphorylation-dependent adduction of tau by carbonyl products resulting from lipid peroxidation creates the neurofibrillary tangle-related antigen, Alz50. To determine whether epitopes of carbonyl-modified tau are major conformational changes associated with neurofibrillary tangle formation, we examined seven distinct antibodies raised against neurofibrillary tangles that recognize unique epitopes of tau in Alzheimer disease. Consistently, all seven antibodies recognize tau more strongly (4- to 34-fold) after treatment of normal tau with the reactive carbonyl, 4-hydroxy-2-nonenal (HNE), but only when tau is in the phosphorylated state. These findings not only support the idea that oxidative stress is involved in neurofibrillary tangle formation occurring in brains of Alzheimer disease patients, but also show, for the first time, that HNE modifications of tau promote and contribute to the generation of the major conformational properties defining neurofibrillary tangles.     

Nonhuman Amyloid Oligomer Epitope Reduces Alzheimer’s-Like Neuropathology in 3xTg-AD Transgenic Mice

Accumulation of beta-amyloid (Aβ) is an important pathological event in Alzheimer’s disease (AD). It is now well known that vaccination against fibrillar Aβ prevents amyloid accumulation and preserves cognitive function in transgenic mouse models. To study the effect of vaccination against generic oligomer epitopes, Aβ oligomers, islet amyloid polypeptide oligomers, random peptide oligomer (3A), and Aβ fibrils were used to vaccinate 3xTg-AD, which develop a progressive accumulation of plaques and cognitive impairment. Subcutaneous administration of these antigens markedly reduced total plaque load (Aβ burden) and improved cognitive function in the 3xTg-AD mouse brains as compared to controls. We demonstrated that vaccination with this nonhuman amyloid oligomer generated high titers of specifically antibodies recognizing Aβ oligomers, which in turn inhibited accumulation of Aβ pathology in mice. In addition to amyloid plaques, another hallmark of AD is tau pathology. It was found that there was a significant decline in the level of hyper-phosphorylated tau following vaccination. We have previously shown that immunization with 3A peptide improves cognitive function and clears amyloid plaques in Tg2576 mice, which provides a novel strategy of AD therapy. Here, we have shown that vaccination with 3A peptide in 3xTg-AD mice not only clears amyloid plaques but also extensively clears abnormal tau in brain.     

Granular tau oligomers as intermediates of tau filaments

Neurofibrillary tangles (NFTs) are pathological hallmarks of several neurodegenerative disorders, including Alzheimer's disease (AD). NFTs are composed of microtubule-binding protein tau, which assembles to form paired helical filaments (PHFs) and straight filaments. Here we show by atomic force microscopy that AD brain tissue and in vitro tau form granular and fibrillar tau aggregates. CD spectral analysis and immunostaining with conformation-dependent antibodies indicated that tau may undergo conformational changes during fibril formation. Enriched granules generated filaments, suggesting that granular tau aggregates may be an intermediate form of tau fibrils. The amount of granular tau aggregates was elevated in prefrontal cortex of Braak stage I cases compared to that of Braak stage 0 cases, suggesting that granular tau aggregation precedes PHF formation. Thus, granular tau aggregates may be a relevant marker for the early diagnosis of tauopathy. Reducing the level of these aggregates may be a promising therapy for tauopathies and for promoting healthy brain aging.     

Alzheimer's Disease Neurofibrillary Tangles Contain Mitosis-Specific Phosphoepitopes

Abstract: Paired helical filaments (PHFs) are the major components of neurofibrillary lesions present in Alzheimer's disease (AD). PHFs are composed of the microtubule-associated protein (MAP) τ, which is abnormally phosphorylated in AD. Normal fetal τ is also phosphorylated and shares certain phosphoepitopes with PHF-τ. The abnormal phosphorylation of PHF-τ is considered to be involved in the formation of PHFs and subsequent degeneration of AD neurons. We have previously shown that other neuronal MAPs, such as MAP1B, contain mitosis-specific phosphoepitopes. In addition to mitotic cells, these epitopes are also expressed in fetal brain and PC12 cells during differentiation and neurite outgrowth. One hypothesis regarding the etiology of AD involves the reactivation of a fetal-like state and mitotic conditions in selected neurons. To determine if similar mitosis-associated phosphoepitopes appeared in AD, sections of hippocampal tissue were stained for immunoreactivity with antibodies recognizing both τ and mitotic phosphoepitopes. Both the MPM2 mitotic phosphoepitope antibody and the AT8 PHF-τ antibody stained neurofibrillary lesions and colocalized to pyramidal neurons in AD samples. In addition, PHFs isolated from an AD brain reacted with both antibodies. The MPM2 antibody specifically reacted with τ in the isolated PHF fraction but not normal adult τ. In addition, MPM2 failed to react with normal fetal or adult τ obtained from rat brains. The MPM2 antibody also recognized human MAP1B; however, MAP1B was not present in the PHF fraction. Our results indicate that MPM2 recognized a phosphoepitope present on PHF-τ. Because normal fetal or adult rat brain τ did not express the MPM2 epitope, it is likely that this phosphoepitope is specific for the disease state.     

Analysis of N-glycans of pathological tau: possible occurrence of aberrant processing of tau in Alzheimer's disease

In a previous study [Wang et al. (1996) Nat. Med. 2, 871-875], Wang et al. found (i) that abnormally hyperphosphorylated tau (AD P-tau) isolated from Alzheimer's disease (AD) brain as paired helical filaments (PHF)-tau and as cytosolic AD P-tau but not tau from normal brain were stained by lectins, and (ii) that on in vitro deglycosylation the PHF untwisted into sheets of thin straight filaments, suggesting that tau only in AD brains is glycosylated. To elucidate the primary structure of N-glycans, we comparatively analyzed the N-glycan structures obtained from PHF-tau and AD P-tau. More than half of N-glycans found in PHF-tau and AD P-tau were different. High mannose-type sugar chains and truncated N-glycans were found in both taus in addition to a small amount of sialylated bi- and triantennary sugar chains. More truncated glycans were richer in PHF-tau than AD P-tau. This enrichment of more truncated glycans in PHF might be involved in promoting the assembly and or stabilizing the pathological fibrils in AD.     

Isoaspartate formation and neurodegeneration in Alzheimer's disease

We reviewed here that protein isomerization is enhanced in amyloid-beta peptides (Abeta) and paired helical filaments (PHFs) purified from Alzheimer's disease (AD) brains. Biochemical analyses revealed that Abeta purified from senile plaques and vascular amyloid are isomerized at Asp-1 and Asp-7. A specific antibody recognizing isoAsp-23 of Abeta further suggested the isomerization of Abeta at Asp-23 in vascular amyloid as well as in the core of senile plaques. Biochemical analyses of purified PHFs also revealed that heterogeneous molecular weight tau contains L-isoaspartate at Asp-193, Asn-381, and Asp-387, indicating a modification, other than phosphorylation, that differentiates between normal tau and PHF tau. Since protein isomerization as L-isoaspartate causes structural changes and functional inactivation, or enhances the aggregation process, this modification is proposed as one of the progression factors in AD. Protein L-isoaspartyl methyltransferase (PIMT) is suggested to play a role in the repair of isomerized proteins containing L-isoaspartate. We show here that PIMT is upregulated in neurodegenerative neurons and colocalizes in neurofibrillary tangles (NFTs) in AD. Taken together with the enhanced protein isomerization in AD brains, it is implicated that the upregulated PIMT may associate with increased protein isomerization in AD. We also reviewed studies on PIMT-deficient mice that confirmed that PIMT plays a physiological role in the repair of isomerized proteins containing L-isoaspartate. The knockout study also suggested that the brain of PIMT-deficient mice manifested neurodegenerative changes concomitant with accumulation of L-isoaspartate. We discuss the pathological implications of protein isomerization in the neurodegeneration found in model mice and AD.     

Immunohistochemical evidence for reorganization of tau in the plaques and tangles in Alzheimer''s dissease

Summary Cytochemical and biochemical techniques have been used to assess the relationship of epitopes on the microtubuleassociated protein, tau, to the cytoskeletal pathology of Alzheimer's disease. The main probes were Tau-1 and Alz-50, two monoclonal antibodies which recognize tau and a potentially related 68kDa protein. Sequential treatment of tissue slices with combinations of the antibodies showed that each blocked the binding of the other to neurofibrillary tangles and neuritic plaques but not to normal axons. Western blot analysis of tau proteins isolated from Alzheimer's disease brains did not reveal such blocking patterns. The issue of steric hindrance affecting antibody binding in tissue sections was addressed by using Alz-50 in combination with Tau-2, another monoclonal antibody recognizing tau on blots and in Alzheimer's disease pathology. Neither antibody blocked the binding of the other to neurofibrillary tangles and neuritic plaques. These data suggest that the Alz-50 and Tau-1 epitopes are selectively organized in the tangles and plaques to be in close proximity which supports the hypothesis that in Alzheimer's disease pathology, tau is modified.     

Tau proteins of Alzheimer paired helical filaments: abnormal phosphorylation of all six brain isoforms.

Preparations of dispersed paired helical filaments (PHFs) from the brains of Alzheimer's disease and Down's syndrome patients display on gels three principal bands corresponding to abnormally modified forms of the microtubule-associated protein tau. Interpretation of the pattern is difficult because there are six tau isoforms in normal brain and phosphorylation changes their mobility. By enzymatic dephosphorylation at high temperature, we have shifted the three abnormal bands obtained from dispersed PHFs to align with the six nonphosphorylated tau isoforms. By using antibodies specific for some of the inserts that distinguish the various isoforms and label PHFs, we have established a correspondence between PHFs, abnormal bands, and isoforms. This identification of isoforms is a necessary step in unravelling the molecular pathogenesis of PHFs.     

Tau-tubulin kinase phosphorylates tau at Ser-208 and Ser-210, sites found in paired helical filament-tau

Hyperphosphorylated tau protein is known to be a major component of the paired helical filaments (PHFs) that accumulate in the brain of Alzheimer's patients. The kinase that phosphorylated Ser-208 and Ser-210 in PHF-tau had remained unknown. We used anti-pS208 and anti-pS210 antibodies and Western blots to confirm that the tau-tubulin kinase (TTK) phosphorylates tau at Ser-208 and at Ser-210. Using partial amino acid sequences of purified bovine brain TTK, a mouse cDNA of TTK was isolated and the sequence was determined. Its 963 bp coding region is composed of 320 amino acids and encodes a 36 kDa protein indistinguishable in size from authentic bovine brain TTK. Our immunoblot analysis demonstrated that TTK is ubiquitously distributed in the rat tissues, and that it is developmentally regulated in the rat brain.     

New Phosphorylation Sites Identified in Hyperphosphorylated Tau (Paired Helical Filament-Tau) from Alzheimer's Disease Brain Using Nanoelectrospray Mass Spectrometry

Abstract: Paired helical filaments (PHFs) are the structural constituents of neurofibrillary tangles in Alzheimer's disease and are composed of hyperphosphorylated forms of the microtubule-associated protein tau (PHF-tau). Pathological hyperphosphorylation of tau is believed to be an important contributor to the destabilisation of microtubules and their subsequent disappearance from tangle-bearing neurons in Alzheimer's disease, making elucidation of the mechanisms that regulate tau phosphorylation an important research goal. Thus, it is essential to identify, preferably by direct sequencing, all of the sites in PHF-tau that are phosphorylated, a task that is incomplete because of the difficulty to date of purifying insoluble PHF-tau to homogeneity and in sufficient quantities for structural analysis. Here we describe the solubilisation of PHF-tau followed by its purification by Mono Q chromatography and reversed-phase HPLC. Phosphopeptides from proteolytically digested PHF-tau were sequenced by nanoelectrospray mass spectrometry. We identified 22 phosphorylation sites in PHF-tau, including five sites not previously identified. The combination of our new data with previous reports shows that PHF-tau can be phosphorylated on at least 25 different sites.     

Abnormal tau proteins from Alzheimer's disease brains. Purification and amino acid analysis

Abnormal tau proteins (PHF-tau) were isolated from Alzheimer's disease brains by treatment of paired helical filament enriched-fractions with perchloric acid and boiling of the acid precipitable fraction with beta-mercaptoethanol. These proteins were purified further by a second perchloric acid treatment. The purified PHF-tau proteins were soluble in buffers devoid of sodium dodecyl sulfate. However, they were similar to the abnormal tau extracted from paired helical filaments with sodium dodecyl sulfate, also named A68, in molecular mass (68, 64, and 60 kDa), isoelectric point (pI 5.5-6.5), reactivity with anti-tau antibodies, and in requirement for alkaline phosphatase treatment to bind the Tau-1 antibody. Compared to normal tau, the soluble PHF-tau contained 100% more glycine and 35% less lysine residue. The results suggest that besides phosphorylation other types of modification may be involved in differentiating PHF-tau from normal tau.     

Tau-66: evidence for a novel tau conformation in Alzheimer's disease

We have characterized a novel monoclonal antibody, Tau-66, raised against recombinant human tau. Immunohistochemistry using Tau-66 reveals a somatic-neuronal stain in the superior temporal gyrus (STG) that is more intense in Alzheimer's disease (AD) brain than in normal brain. In hippocampus, Tau-66 yields a pattern similar to STG, except that neurofibrillary lesions are preferentially stained if present. In mild AD cases, Tau-66 stains plaques lacking obvious dystrophic neurites (termed herein 'diffuse reticulated plaques') in STG and the hippocampus. Enzyme-linked immunosorbent assay (ELISA) analysis reveals that Tau-66 is specific for tau, as there is no cross-reactivity with MAP2, tubulin, Abeta(1-40), or Abeta(1-42), although Tau-66 fails to react with tau or any other polypeptide on western blots. The epitope of Tau-66, as assessed by ELISA testing of tau deletion mutants, appears discontinuous, requiring residues 155-244 and 305-314. Tau-66 reactivity exhibits buffer and temperature sensitivity in an ELISA format and is readily abolished by SDS treatment. Taken together these lines of evidence indicate that the Tau-66 epitope is conformation-dependent, perhaps involving a close interaction of the proline-rich and the third microtubule-binding regions. This is the first indication that tau can undergo this novel folding event and that this conformation of tau is involved in AD pathology.     

Stress-induced hyperphosphorylation of tau in the mouse brain

We previously showed that starvation causes reversible hyperphosphorylation of tau in the mouse brain. To explore possible involvement of stress in tau hyperphosphorylation quantitative analysis of phosphorylated tau in four brain regions of mice subjected to cold water stress (CWS) was made by immunoblot analyses using phosphorylation-dependent antibodies directed to eight sites on tau known to be hyperphosphorylated in the brain of Alzheimer's disease (AD) patients. Ser199, Ser202/Thr205, Thr231/Ser235 were hyperphosphorylated 20 and 40 min after CWS. The response was pronounced in the hippocampus and cerebral hemisphere, but weak in the cerebellum in parallel with the regional vulnerability in AD. Among the regulatory phosphorylation of protein kinases studied, a transient phosphorylation of tau protein kinase I/glycogen synthase kinase 3beta at Ser9 was most conspicuous.     

Two-Dimensional Characterization of Paired Helical Filament-Tau from Alzheimer's Disease: Demonstration of an Additional 74-kDa Component and Age-Related Biochemical Modifications

Abstract: PHF-tau proteins are the major components of the paired helical filament (PHF) from Alzheimer's disease (AD) neurofibrillary lesions. They differ both qualitatively and quantitatively in their degree of phosphorylation when compared with native tau proteins. However, little is known about the extent and heterogeneity of phosphorylated sites or the isoform composition and the isoelectric variants of PHF-tau. Therefore, we have characterized PHF-tau proteins from cortical brain tissue homogenates of 13 AD patients using two-dimensional gel electrophoresis. Whatever the topographical origin of brain tissue homogenates, PHF-tau proteins shared the same two-dimensional gel electrophoresis profile made of a tau triplet of 55, 64, and 69 kDa. A 74-kDa hyperphosphorylated tau component was detected particularly in the youngest and most severely affected AD patients. This additional component of hyperphosphorylated tau was shown to correspond to the longest brain tau isoform. Furthermore, the isoelectric points of PHF-tau from older AD patients were significantly more basic, indicating a lower degree of phosphorylation. These results show that the severity of neurofibrillary degeneration of AD is modulated by age.