Phosphorylation and cleavage of tau in non-AD tauopathies

The tau protein, well known as the primary component of neurofibrillary tangles, also comprises the Pick bodies found in Pick’s disease (PiD) and the glial lesions associated with progressive supranuclear palsy (PSP) and cortico-basal ganglionic degeneration (CBD). Many of the tau alterations that are characteristic of Alzheimer’s disease have also been identified in PSP and CBD. In this report, we examine three non-AD tauopathies (PSP, CBD, and PiD) for the presence of two specific tau alterations, phosphorylation at Ser422 and truncation at Asp421. We find that truncation at Asp421 is an alteration that is unique to neuronal lesions, occurring in Pick bodies as well as in neurofibrillary tangles, but not in lesions associated with glia. Conversely, phosphorylation at Ser422 is not only present in all these lesions, but identifies additional glial and neuronal pathology in disease-susceptible cortical regions. These results suggest that the molecular alterations of tau that occur during the initial process of tangle formation in AD are similar in non-AD tauopathies, but the middle and later changes are not common to all diseases. This work is supported by grants AG021184 and AG09466 from the NIH.     

Earliest stages of tau conformational changes are related to the appearance of a sequence of specific phospho-dependent tau epitopes in Alzheimer's disease

Neurofibrillary tangles (NFT) and dystrophic neurites represent dense cytoplasmic accumulations of abnormal polymers in the brain of patients with Alzheimer's disease (AD). These polymers are referred to as paired helical filaments (PHFs) whose main structural core is composed of tau protein. Tau processing has been associated with hyperphosphorylation and truncation that results in PHF assembly. Both molecular events appear to cause conformational change of tau molecules [11,17,32]. In this regard, in a previous work focused on the analysis of patterns of immunolabeling in pre-tangle cells, we found that regional changes precede the structural modifications in tau [32]. In the present study, we further analyzed the early stages of tau processing in pre-tangle cells by using a variety of immunological markers of specific N-terminus phosphorylation tau sites. We used AT100, TG-3, AT8, pT231, Alz-50, Tau-C3 and 423 antibodies that recognize different abnormal tau epitopes in AD brains. These antibodies were combined and analyzed using a confocal microscope. Our results indicate that the early stages of abnormal tau processing are characterized by a sequential appearance of specific phospho-dependent epitope. The cascade of appearance of the antibodies is: pT231 --> TG-3 --> AT8 -->AT100 --> Alz-50. In addition; truncation at Asp-421 of the C-terminus of tau protein, as detected by Tau-C3, is also an early molecular event in tau protein aggregation prior to PHF formation in AD.     

Cellular tau pathology and immunohistochemical study of tau isoforms in sporadic tauopathies

Pathological inclusions in neurons and glial cells containing fibrillary aggregates of abnormally hyperphosphorylated tau protein are characteristic features in sporadic tauopathies. In the first part of this paper we outline the morphological features of some major sporadic tauopathies. In the second part, to better define the tau isoform composition, we report on the immunohistochemistry of tau isoforms in autopsied brains, including two cases with AD, two with diffuse neurofibrillary tangles with calcification, four with Pick’s disease with Pick bodies (PiD), seven with progressive supranuclear palsy (PSP), six with corticobasal degeneration (CBD) and seven cases with argyrophilic grain disease. We used two monoclonal antibodies, RD3 and RD4, and a polyclonal antibody for exon 10 that effectively distinguish between three‐repeat (3R) tau and four‐repeat (4R) tau. Neuronal neurofibrillary tangles (NFT) in AD and diffuse neurofibrillary tangles with calcification contained both 3R‐tau and 4R‐tau. The Pick bodies were immunopositive for 3R‐tau in two cases; however, in two other cases they were mainly immunopositive for 4R‐tau. Thus, Pick bodies demonstrated heterogeneity. 3R‐tau PiD contained 3R‐tau glial inclusions, and 4R‐tau PiD contained mainly 4R‐tau glial inclusions. Glial inclusions were more abundant in 4R‐tau PiD cases. In progressive supranuclear palsy and CBD, both neuronal and glial tau accumulation forming NFT, pretangles, tuft‐shaped astrocytes, astrocytic plaques, coiled bodies and threads demonstrated 4R‐tau in the cerebral cortices, although in the basal ganglia and brainstem neuronal and glial inclusions were occasionally immunopositive for 3R‐tau in addition to 4R‐tau. Argyrophilic grains (AG) were immunopositive for 4R‐tau, although pretangles were weakly stained for 4R‐tau. Thus the immunoreactivity for 4R‐tau was different between AG and pretangles. Therefore, the isoform composition on immunohistochemical study showed heterogeneity in PiD, and was not uniform in the basal ganglia and brain stem in PSP and CBD. It is suggested that the isoform composition of sporadic tauopathies may have a spectrum in individual cases, and cellular isoform composition may differ in various brain regions.     

Accumulation of aspartic acid421- and glutamic acid391-cleaved tau in neurofibrillary tangles correlates with progression in Alzheimer disease

Truncations of tau protein at aspartic acid421 (D421) and glutamic acid391 (E391) residues are associated with neurofibrillary tangles (NFTs) in the brains of Alzheimer disease (AD) patients. Using immunohistochemistry with antibodies to D421- and E391-truncated tau (Tau-C3 and MN423, respectively), we correlated the presence of NFTs composed of these truncated tau proteins with clinical and neuropathologic parameters in 17 AD and 23 non-AD control brains. The densities of NFTs composed of D421- or E391-truncated tau correlated with clinical dementia index and Braak staging in AD. Glutamic acid391 tau truncation was prominent in the entorhinal cortex, whereas D421 truncation was prominent in the subiculum, suggesting that NFTs composed of either D421- or E391-truncated tau may be formed mutually exclusively in these areas. Both truncations were associated with the prevalence of the apolipoprotein E epsilon4 allele. By double labeling, intact tau in NFTs was commonly associated with D421-cleaved tau but not with E391-truncated tau; D421-cleaved tau was never associated with E391-truncated tau. These results indicate that tau is not randomly proteolyzed at different domains, and that proteolysis occurs sequentially from the C-terminus to inner regions of tau in AD progression. Identification of NFTs composed of tau at different stages of truncation may facilitate assessment of neurofibrillary pathology in AD.     

Alz-50 recognizes a phosphorylated epitope of tau protein.

Alz-50 is a monoclonal antibody that detects antigens enriched in the brain tissue of Alzheimer's disease (AD) patients. Although Alz-50 recognizes tau, an identified integral constituent of the AD paired helical filament (PHF), the exact nature of the antigenic site is unknown. An immunoblot analysis demonstrated that the antigenic sites to Alz-50 are diminished by acid phosphatase treatment. Consistent with this finding, Alz-50 antigens were more concentrated in brain homogenates prepared with phosphatase inhibitors. The epitope in tau with which Alz-50 reacts is located in the carboxy terminus within a 14-amino acid region from just beyond the microtubule-binding repeats to the carboxy terminus. An isolated carboxy-terminal chymotryptic peptide from bovine brain tau reactive with Alz-50 was analyzed by fast-atom-bombardment mass spectroscopy (FAB-MS) and was found to be present as both a monophosphopeptide and a nonphosphorylated peptide. The immunohistological analysis has demonstrated that Alz-50 staining of neurofibrillary tangles (NFTs) is sensitive to acid phosphatase but not to alkaline phosphatase. Furthermore, Alz-50 staining of NFTs was effectively adsorbed by a high concentration of phosphoserine but not by serine or phosphothreonine. These results strongly suggest that Alz-50 recognizes a phosphorylated epitope in the carboxy terminus of tau which has not been previously detected by using alkaline phosphatase. The strong Alz-50 staining in AD samples may represent another association between a phosphorylation state and neurofibrillary lesions. As a marker of the inchoate tangle-bearing neuron, the characterization of the Alz-50 epitope in tau offers a partial molecular basis for the modifications that contribute to the assembly of PHFs.     

Epitope expression and hyperphosphorylation of tau protein in corticobasal degeneration: differentiation from progressive supranuclear palsy

Corticobasal degeneration (CBD) is a rare, progressive neurological disorder characterized by widespread neuronal and glial accumulation of abnormal tau protein. Using immunohistochemistry we analyzed tau epitope expression and phosphorylation state in CBD and compared them to cytoskeletal changes in Alzheimer's disease (AD) and progressive supranuclear palsy (PSP). Epitopes spanning the entire length of the tau protein were present in CBD inclusions. An antibody against the alternatively spliced exon 3 did not recognize cytoskeletal lesions in CBD, but did in AD and PSP. Tau epitopes from each region of the molecule were present in cytoskeletal inclusions in CBD, including gray matter astrocytic plaques, gray and white matter threads, and oligodendroglial inclusions. As in AD, tau from CBD was highly phosphorylated. Antibodies that recognized phosphorylated tau epitopes reacted with material from CBD in a highly phosphatase-dependent manner. Again, all types of inclusions contained phosphorylated epitopes. We conclude that abnormal tau protein in CBD comprises the entire tau molecule and is highly phosphorylated, but is distinguished from AD and PSP by the paucity of epitopes contained in the alternatively spliced exon 3.     

Phosphorylated serine422 on tau proteins is a pathological epitope found in several diseases with neurofibrillary degeneration

Neuronal inclusions with bundles of abnormal filaments made of tau polymers are found in numerous diseases with neurofibrillary degeneration. Tau proteins are the basic components of paired helical filaments (PHF) in Alzheimer’s disease (AD), and are abnormally phosphorylated. A disease-specific phosphorylation site at serine422 was demonstrated on PHF, but not on tau proteins from biopsy-derived brain samples. In the present study, we report the characterization of a polyclonal antibody (988) against the serine422 phosphorylation site. By using biochemical and immunohistochemical methods, we confirmed that it is not found on tau proteins from biopsy- or autopsy-derived control samples, and we investigated the presence of this epitope on tau proteins in several neurodegenerative disorders, including AD, Down syndrome (DS), Guamanian amyotrophic lateral sclerosis/Parkinsonism-dementia complex (ALS/PDC), corticobasal degeneration (CBD), progressive supranuclear palsy (PSP), postencephalitic parkinsonism (PEP) and Pick’s disease (PiD). By Western blotting, antibody 988 labeled the characteristic tau triplet (tau 55, 64, 69) in AD, DS, Guamanian ALS/PDC and PEP. PSP and CBD exhibited their typical tau doublet (tau 64, 69), whereas the doublet tau 55 and 64 was detected in PiD. In all of these neurodegenerative disorders, antibody 988 clearly labeled NFT and dystrophic neurites, as well as Pick bodies in PiD cases, whereas no staining was observed in control cases. These data indicate that phosphorylation of serine422 on tau proteins is a common feature among neurodegenerative disorders and is therefore not specific of AD. Moreover, phosphorylation of this epitope permits the distinction between normal tau proteins and pathological tau proteins. Received: 24 July 1998 / Revised: 8 September 1998 / Accepted: 14 September 1998     

Compartmentalized tau hyperphosphorylation and increased levels of kinases in transgenic mice.

The formation of neurofibrillary tangles in Alzheimer's disease is preceded by a pretangle stage of hyperphosphorylated tau. To characterize pretangle tau in vivo, we correlated, in human tau transgenic mice, levels of kinases known to phosphorylate tau in vitro with the phosphorylation of tau at specific epitopes. Levels of cyclin-dependent kinase-5 were increased in axons of CA1 pyramidal neurons, where tau was phosphorylated specifically at the AD2 epitope Ser396/Ser404. The 12E8 epitope serine262/serine356 and the AT180 epitope threonine231/serine235 were phosphorylated in dendrites, and colocalized with increased levels of glycogen synthase kinase-3. CA1 neurons phosphorylated tau at more epitopes than dentate gyrus neurons, suggesting that tau phosphorylation is cell type-specific, a possible explanation for the spatial distribution of neurofibrillary tangles.     

Microtubule assembly competence analysis of freshly-biopsied human tau,dephosphorylated tau,and Alzheimer tau

Phosphorylation of the microtubule-associated protein tau regulates its binding to microtubules; highly phosphorylated tau is also a prime component of paired helical filaments (PHFs) of Alzheimer's disease (AD). Tau from freshly biopsied human, monkey, and rat brain share similar electrophoretic mobility patterns and overlapping phosphorylated epitopes when compared to AD tau isolated from AD brain. We compared the microtubule reassembly competence of fresh isolates of phosphorylated tau to that of maximally dephosphorylated tau and tau from AD brain. A rapid procedure was developed which permitted the enrichment of phosphorylated and dephosphorylated tau from human biopsies in the absence of protein kinase and phosphatase activity. Microtubule assembly assays, using a spectrophotometric measure and purified bovine brain tubulin, were used to correlate assembly competence with states of tau electrophoretic mobility. Maximally dephosphorylated human biopsy-derived tau and monkey tau were assembly competent; tau from AD brain was virtually unable to direct microtubule assembly. Unmodified, biopsy-derived tau from non-AD brain was intermediate in assembly competence relative to AD tau and dephosphorylated tau. Several lines of evidence were used to correlate phosphorylation states of tau with microtubule assembly. First, in vitro dephosphorylation of human biopsy-derived tau with either PP2A or PP2B alone or in combination led to increasing assembly competence as the electrophoretic mobility of tau increased. Second, addition of the protein phosphatase inhibitor okadaic acid (10 μM) to brain-slice preparations slowed electrophoretic mobility of tau and decreased binding competence. We suggest that tau derived from freshly-biopsied brain exists in a range of phosphorylated states, and that dephosphorylation by PP2A and/or PP2B increases microtubules assembly competence. © 1996 Wiley-Liss, Inc.     

The extent of phosphorylation of fetal tau is comparable to that of PHF-tau from Alzheimer paired helical filaments

The relationship between Alzheimer's disease (AD) and expression of fetal proteins was examined by: (i) determining the phosphate content of tau prepared from fetal brains (F-tau); (ii) comparing F-tau, tau from normal adult human brains (N-tau) and tau from paired helical filaments in AD brains (PHF-tau) for phosphate content; and (iii) testing the reactivity of F-tau with five antibodies known to recognize PHF-tau. The antibodies have been reported to recognize phosphate dependent epitopes at the carboxy-terminal half of the tau molecule. Our data shows that on the average, F-tau contains 7 mol phosphate/mol protein, which is comparable to the phosphate content of PHF-tau, but is 3–4 times higher than that of N-tau. Immunoblotting shows that all of the tested antibodies reacted with F-tau on immunoblots, indicating that F-tau and PHF-tau are phosphorylated at similar sites. A difference between PHF-tau and F-tau is the state of phosphorylation in the Tau-1 epitope, an epitope reactive with a monoclonal anti-tau antibody, Tau-1. This epitope, which is phosphorylated in all PHF-tau, is phosphorylated only in some of the F-tau. The sharing of phosphorylated sites between F-tau and PHF-tau has also been reported by others in studies with antibodies to different and similar phosphorylated epitopes. Together these observations indicate that the extent and the site of phosphorylation in F-tau and PHF-tau tau are similar. Although hyperphosphorylation of tau proteins may be an important step for PHF formation, the absence of AD type pathology in fetal brains containing hyperphosphorylated tau suggests that the transformation of soluble forms of normal tau to AD type cytoskeletal abnormalities may require the presence of other factors.     

DC11: a novel monoclonal antibody revealing Alzheimer's disease-specific tau epitope

Using tau protein extracts from Alzheimer's disease (AD) brain tissue, we generated a monoclonal antibody (mAb DC11) which decorated neurofibrillary pathology in brain derived from AD patients on immunohistochemistry, and which lacked reactivity with healthy brain tissue. The same pattern of DC11 specificity was observed on Western blot. The main constituent of structures decorated by DC11 is microtubule-associated protein tau. In Western blot, DC11 recognized neither native healthy tau nor its full length recombinant counterpart. However, the mAb showed strong immunoreactivity with truncated tau (residues tau151-421), thus indicating the requirement for a conformational epitope. Importantly, the DC11 epitope was phosphorylation independent. The immunochemical parameters of mAb show that DC11 could represent a novel structural probe with the specificity for conformation of pathological tau present in AD brains.     

Regional conformational change involving phosphorylation of tau protein at the Thr231, precedes the structural change detected by Alz-50 antibody in Alzheimer's disease

Neurofibrillary tangles (NFTs) are the neuropathological hallmarks in Alzheimer's disease (AD). Densities of NFTs correlate with the dementia status. NFTs reflect the intracellular accumulation of abnormal paired helical filaments (PHFs) composed of the microtubule-associated protein tau. Hyperphosphorylation and truncation have been proposed as key events leading to the genesis of PHFs. A recent hypothesis involving conformational changes has been emerging. These structural modifications of the tau protein were detected by monoclonal antibodies (mAbs) recognizing discontinuous epitopes along the tau molecule such as Alz-50, Tau-66 and MC1. A new mAb, TG-3, detects an early pathology in AD. The epitope of mAb TG-3 maps to phosphorylated Thr231 when the tau molecule is conformationally altered. In the present study, we used confocal microscopy to analyze the state of tau molecule adopting the TG-3 conformation during tangle formation. We also compared mAb TG-3 immunoreactivity with that of mAb Alz-50. Immunoelectronmicroscopy was also performed. N- and C- termini markers evidenced that the tau molecule is intact when it adopts the TG-3 conformation. In addition to NFT, mAb TG-3 also recognized NFT-not bearing-neurons suggesting an early processing of tau prior to NFT formation. Ultrastructural analysis evidenced the presence of TG-3 and Alz-50 immunoreactive products on organelles including mitochondria and endoplasmic reticulum. Nuclear heterochromatin was densely immunolabelled. These results together with the fact that TG-3 immunoreactivity is related to intact tau suggest that the conformation recognized by TG-3 is early staged in the neuronal pathology of AD. In addition, we document that the earliest changes in tau occur closely associated with organelles and heterochromatin.     

Small heat shock proteins Hsp27 or alphaB-crystallin and the protein components of neurofibrillary tangles: tau and neurofilaments

The heat-shock proteins (HSPs) Hsp27 and alphaB-crystallin are up-regulated in Alzheimer's disease (AD), but the extent of this and the consequences are still largely unknown. The HSPs are involved in protein degradation and protection against protein aggregation, and they interact with several cytoskeletal components such as microtubules (MT) and neurofilaments (NF). AD pathology includes aggregated proteins (tau, NF), decreased protein degradation, and cytoskeletal disruption. It is thus of interest to investigate more closely the possible roles of the HSPs in AD pathology. The expressions of Hsp27 and alphaB-crystallin in AD brain samples were significantly increased (by approximately 20% and approximately 30%, respectively) and correlated significantly with phosphorylated tau and NF proteins. To investigate the consequences of increased HSP levels on tau and NF regulation, N2a cells were transfected with Hsp27 or alphaB-crystallin constructs, and overexpression of the HSPs was confirmed in the cells. Increased tau phosphorylation at the Ser262 site in the N2a cells was regulated by Hsp27 overexpression (possibly through p70S6k), whereas the overexpression of alphaB-crystallin resulted in decreased levels of phosphorylated tau, NF, and GSK-3beta. It was also shown that overexpression of HSPs causes an increase in the percentage of cells present in the G(1) phase. The results presented suggest that a cellular defense against dysregulated proteins, in the form of Hsp27 and alphaB-crystallin, might contribute to the cell cycle reentry seen in AD cells. Furthermore, Hsp27 might also be involved in AD pathology by aggravating MT disruption by tau phosphorylation.     

Inhibition of proteasome and Shaggy/Glycogen synthase kinase-3beta kinase prevents clearance of phosphorylated tau in Drosophila

Tauopathies, including Alzheimer's disease (AD), are a group of neurodegenerative disorders characterized by the presence of intraneuronal filamentous inclusions of abnormally phosphorylated tau protein. In AD brains, it has been shown that the level of abnormally phosphorylated tau is higher than in age-matched control brains, suggesting that abnormally phosphorylated tau is resistant to degradation. By using a Drosophila model of tauopathy, we studied the relationship between tau phosphorylation and degradation. We showed that in vivo reduction of proteasome activity results in an accumulation of high-molecular-weight forms of hyperphosphorylated tau. We also found that glycogen synthase kinase (GSK)-3beta-mediated hyperphosphorylated forms of tau are degradable by the proteasomal machinery. Unexpectedly, GSK-3beta inactivation resulted in a very large accumulation of high-molecular-weight species consisting of hyperphosphorylated tau, suggesting that, depending on the kinase(s) involved, tau phosphorylation state affects its degradation differently. We thus propose a model for tauopathies in which, depending on toxic challenges (e.g., oxidative stress, exposure to amyloid peptide, etc.), abnormal phosphorylation of tau by kinases distinct from GSK-3beta leads to progressive accumulation of hyperphosphorylated tau oligomers that are resistant to degradation.     

Anti-tau phospho-specific Ser262 antibody recognizes a variety of abnormal hyper-phosphorylated tau deposits in tauopathies including Pick bodies and argyrophilic grains

The rabbit polyclonal anti-tau phospho-specific Ser262 antibody (577814 Calbiochem) recognizes disease-specific band patterns on Western blots of sarkosyl-insoluble fractions in Alzheimer's disease (AD), progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), argyrophilic grain disease (AGD) and Pick's disease (PiD): four bands of 74/72, 68, 64 and 60 kDa in AD, two bands of 68 and 64 kDa in PSP, CBD and AGD, and two bands of 64 and 60 kDa in PiD. Moreover, anti-tau phospho-specific Ser262 decorates neurons with neurofibrillary tangles, neurons with pre-tangles, dystrophic neurites of senile plaques, neuropil threads, Pick bodies, argyrophilic grains, and coiled bodies. Achromatic neurons in CBD, ballooned neurons in AGD, tufted astrocytes in PSP, astrocytic plaques in CBD and tau-containing astrocytes in AGD are not immunostained with the anti-tau phospho-specific Ser262 antibody. The lack of phospho-specific Ser262 immunoreactivity in tau-containing inclusions in astrocytes suggests different kinase equipment and activation in comparing neurons and astrocytes in tauopathies. Pick bodies in PiD and grains in AGD are weakly, or not all, immunostained in tissue samples with long post-mortem delays, although Ser262 is preserved in brain homogenates corresponding to the same time points processed for Western blot. This indicates postmortem modifications of tau in Pick bodies and argyrophilic grains, but not in other tau-containing inclusions, including paired helical filaments and coiled bodies, and suggests differences in tau conformation, particularly that involving phospho-tau Ser262 among tauopathies. However, it is important to note that phosphorylation of tau at Ser262 does occur in Pick bodies and argyrophilic grains, and this may have important consequences in reducing the capacity of binding phospho-tau to microtubules in these inclusions.     

Phosphorylated serine 199 of microtubule-associated protein tau is a neuronal epitope abundantly expressed in youth and an early marker of tau pathology

Microtubule-associated protein tau is abnormally phosphorylated in many neurodegenerative disorders, and is the major component of neurofibrillary degeneration, a degenerating process with many biochemical phenotypes. The serine 199 (S199) residue of tau is phosphorylated at early and late stages of Alzheimer's disease (AD). We studied the immunohistochemical distribution of this phosphorylated epitope in AD and other neurodegenerative disorders, as well as in controls of different ages. The phosphorylated S199 (S199P) epitope was observed in tau lesions from numerous diseases with neurofibrillary degeneration. This epitope was found to be abundantly expressed in the hippocampus formation in childhood and in young adult brain samples, and more specifically in subsets of neurons vulnerable to neurodegeneration. Interestingly, our data suggests that S199P is particularly resistant to phosphatase activity occurring during post-mortem delays. We suggest a peculiar and important role of the S199 residue as a qualitative indicator of the normal and pathological phosphorylation status of tau proteins.     

Sequence requirements for formation of conformational variants of tau similar to those found in Alzheimer's disease

Alz-50 and MC-1 monoclonal antibody reactivity is dependent on both the extreme N-terminus of tau (residues 7-9) and a 30-amino acid sequence of tau (amino acids 312-342) in the third microtubule binding domain, suggesting that the specificity of the Alz-50 and MC-1 antibodies for Alzheimer's disease (AD) pathological tau lies in their ability to recognize a specific conformation of the tau molecule in AD. The present study uses deletional and site-directed mutants of tau to further refine the C-terminal (third microtubule binding domain) epitope requirements for Alz-50, MC-1, and several new antibodies that recognize similar epitopes in tau to amino acids 313-322 of tau, and to demonstrate that intervening portions of the tau molecule are not required for the formation of conformational variants of tau similar to those seen in AD. Further analysis of deletional and site-directed mutations of tau demonstrate subtle variations in the epitope requirements for Alz-50, MC-1, CP-1, CP-2, and CP-28, suggesting that these antibodies, albeit different, all recognize a similar pathological conformation of tau. Additional experiments using synthetic peptides demonstrate that the NH2-terminal (amino acids 1-18) and COOH-terminal (amino acids 309-326) portions of the Alz-50, MC-1, CP-1, CP-2, and CP-28 epitopes can interact with high affinity under near physiological conditions.     

Endoplasmic reticulum stress induces tau pathology and forms a vicious cycle: implication in Alzheimer's disease pathogenesis

Accumulation of unfolded proteins can disturb the functions of the endoplasmic reticulum (ER), leading to ER-stress or unfolded protein response (UPR). Recent data have shown that activation of UPR can be found in postmortem brains of Alzheimer's disease (AD) patients; and biological markers for activation of UPR are abundant in neurons with diffuse phosphorylated tau. Although these observations suggest a linkage between ER-stress and tau pathology, little is known of their relationship. In this study, we found that high levels of phosphorylated PKR-like ER-resident kinase (p-PERK) and phosphorylated eukaryotic initiation factor 2 alpha (p-eIF2α) as markers for activation of UPR in the hippocampus of aged P301L mutant tau transgenic mice. The immunoreactivity of p-PERK was found to co-localize with that of phosphorylated tau. We then hypothesized that phosphorylation of tau could induce ER-stress and vice versa in promoting AD-like pathogenesis. By using the protein phosphatase 2A inhibitor okadaic acid (OA) as an inducer for phosphorylation of tau, we found that primary cultures of rat cortical neurons treated with OA triggered UPR as indicated by increased levels of p-PERK and p-eIF2α, splicing of mRNA for xbp-1 and elevated levels of mRNA for GADD153. On the other hand, thapsigargin as an ER-stress inducer stimulated phosphorylation of tau at Thr231, Ser262 and Ser396. Thapsigargin also induced activation of caspase-3 and cleavage of tau. These findings suggested that ER-stress and hyperphosphorylation of tau could be induced by each other to form a vicious cycle to propagate AD-like neurodegeneration.     

Specific tau phosphorylation sites in hippocampus correlate with impairment of step-down inhibitory avoidance task in rats

Microtubule associated protein tau is abnormally phosphorylated in Alzheimer's disease (AD) and aggregates as paired helical filaments (PHFs) in neurofibrillary tangles (NFTs), which are one of the pathological signatures of AD and their presence correlates with severity of dementia. Dysfunction of protein phosphatases in the effected neurons is proposed to be a possible causative factor to AD development. We show here that the pattern of tau phosphorylation correlates with the decline of memory retention ability in rat brain. In our study, we have chosen 55 rats of full age to conduct the discrimination between their normal and low ability of memory retention in one-trial step-down test. It was found that among rats that developed the impairment in memory retention in step-down inhibitory avoidance task, tau protein in their hippocampus was hyperphosohorylated at Thr231/Ser235 (M4) sites of tau, and the significantly increased expression of PP-1 and the decreased one of PP-2B were also determined by Western blot and/or immunohistochemistry. It is implicated that: (1) the hyperphosphorylation of tau at M4 sites may be crucial to affect the memory retention of elder rats; (2) PP-1 might participate in the regulation of phosphorylation at Thr231 and Ser235 epitope of tau in vivo, and the up-regulation of PP-1 content could be in relation to tau hyperphosphorylation at Thr231/Ser235 sites of brain tau and the worse memory retention of rats indirectly; and (3) the decline of PP-2B content could induced the hyperphosphorylation of tau at M4 sides in vivo.