Production and characterization of a monospecific antiserum (A128) to disaggregated Alzheimer paired helical filaments

Paired helical filaments (PHF), which constitute neurofibrillary tangles (NFT) and neuritic plaque (NP) neurites, serve as a useful marker for Alzheimer disease (AD). We have isolated AD PHF in a highly purified and disaggregated form for use as an immunogen to produce a heterologous polyclonal antiserum in rabbits. One rabbit was maintained long-term for the high quality of the antiserum it produced. Through absorptions with normal brain tissue, we were able to produce a monospecific antiserum which reacts only with NFT and NP neurites in AD brain tissue sections. We further demonstrated the specificity of this antiserum by electron microscopic immunohistochemistry, gel diffusion analysis, and immunoblotting. This antiserum also showed immunoreactivity to NFT of Down syndrome and progressive supranuclear palsy, and to the Pick bodies of Pick disease, but not to the Lewy bodies of idiopathic Parkinson disease. This well-characterized antiserum, all from one rabbit, offers several unique advantages to the study of the nature, origin, and interrelationships of filamentous protein abnormalities in AD and other neurodegenerative disorders.     

Alzheimer's disease: microtubule-associated proteins 2 (MAP 2) are not components of paired helical filaments

In Alzheimer's disease, the most characteristic neuropathological changes are the formation of neurofibrillary tangles (NFT) and neuritic plaques (NP) characterized by the presence of bundles of paired helical filaments (PHF) that accumulate in the degenerating neurites and neuronal cell bodies. Although the protein composition of the PHF is ill-defined, a number of microtubule-associated proteins have been implicated in these lesions. Here we report results with an antiserum monospecific for the microtubule-associated protein MAP 2 which does not cross-react with any other microtubular protein. Immunostaining with this antibody of sections from an Alzheimer's brain show a strong reactivity with NFT but no reactivity at the level of the NP. On the other hand, immunostaining of Alzheimer's brain sections with another antibody specific for the microtubule-associated protein tau shows strong staining of PHF on both NFT and NP. These findings confirm the presence of the tau proteins in the PHF and strongly suggest that MAP 2 may not be a main structural component of the PHF. Labelling of NFT with the anti-MAP 2 antiserum suggests a non-specific binding of MAP 2 to the PHF during the process of NFT formation.     

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

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

The carboxyl third of tau is tightly bound to paired helical filaments

To obtain definitive evidence that tau is a component of paired helical filaments (PHF) in Alzheimer's disease, we fractionated and sequenced PHF-derived peptides according to a previously described procedure. In the PHF digest, we found four independent tau peptides that were located in the carboxyl third of tau. Subsequent extensive analysis of the PHF digest did not provide any other tau peptides. The conventional PHF antiserum and a new antiserum directed toward formic acid-denatured PHF reacted with the distinct CNBr fragments of tau localized on the carboxy-terminal portion of tau by protein sequencing. From these observations, we conclude that the carboxyl third of tau is tightly bound to PHF.     

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.     

Intrinsically Disordered Proteins in the Neurodegenerative Processes: Formation of Tau Protein Paired Helical Filaments and Their Analysis

1. Several intrinsically disordered proteins (IDPs) play principal role in the neurodegenerative processes of various types. Among them, α-synuclein is involved in Parkinson's disease, prion protein in transmissible spongiform encephalopathies, and tau protein in Alzheimer's disease (AD) and related tauopathies. Neuronal damage in AD is accompanied by the presence of tau protein fibrils composed of paired helical filaments (PHF).2. Tau protein represents a typical IDP. IDPs do not exhibit any stable secondary structure in the free form, but they are able to fold after binding to targets and contain regions with large propensity to adopt a defined type of secondary structure. Binding–folding event at tau protein leading to PHF generation is believed to happen in the course of tauopathies.3. Detailed molecular topology of PHF formation is unknown. There are evidences about the cross-beta structure in PHF core; however the precise arrangement of the tau polypeptide chain is unclear. In this review we summarize current attempts at in vitro PHF reconstruction and the development of methods for PHF structure determination. The emphasis is put on the monoclonal antibodies used as structural molecular probes for research on the role of IDPs in pathogenesis of neurodegenerative diseases.Dedicated to the late Peter Kontsek.     

Hierarchical Phosphorylation of Recombinant Tau by the Paired-Helical Filament-Associated Protein Kinase Is Dependent on Cyclic AMP-Dependent Protein Kinase

Abstract : Immunoaffinity-purified paired helical filaments (PHFs) from Alzheimer's disease (AD) brain homogenates contain an associated protein kinase activity that is able to induce the phosphorylation of PHF proteins on addition of exogenous MgCl₂ and ATP. PHF kinase activity is shown to be present in immunoaffinity-purified PHFs from both sporadic and familial AD, Down's syndrome, and Pick's disease but not from normal brain homogenates. Although initial studies failed to show that the kinase was able to induce the phosphorylation of tau, additional studies presented in this article show that only cyclic AMP-dependent protein kinase-pretreated recombinant tau is a substrate for the PHF kinase activity. Deletional mutagenesis, phosphopeptide mapping, and site-directed mutagenesis have identified the PHF kinase phosphorylation sites as amino acids Thr³⁶¹ and Ser⁴¹² in htau40. In addition, the cyclic AMP-dependent protein kinase phosphorylation sites that direct the PHF kinase have been mapped to amino acids Ser³⁵⁶ and Ser⁴⁰⁹ in htau40. Additional data demonstrate that these hierarchical phosphorylations in the extreme C terminus of tau allow for the incorporation of recombinant tau into exogenously added AD-derived PHFs, providing evidence that certain unique phosphorylations of tau may play a role in the pathogenesis of neurofibrillary pathology in AD.     

One of the antigenic determinants of paired helical filaments is related to tau protein

Paired helical filaments (PHF) are unusual neuronal fibers which accumulate progressively in the brain in Alzheimer's disease (AD). The insolubility of PHF in various kinds of solvents enabled us to obtain highly purified PHF, but prevented the application of conventional analytical methods to identify their components. Here we report that antibodies against purified PHF recognize tau protein, a brain-specific microtubule-associated protein, suggesting that a portion of PHF is tau protein.     

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.     

PHD finger protein 8蛋白多克隆抗体的制备、纯化与检测

PHD finger8(PHF8)蛋白是最新发现的一种带有PHD结构域和Jmjc结构域的蛋白。现有研究表明其可能在基因转录、组蛋白去甲基化等方面发挥重要作用。为研究其功能,本研究构建原核表达载体pET41b-PHF8(aa886-936),在大肠杆菌Escherichia coli BL21中诱导表达带有GST标签的PHF8(aa886-936)亲水片段融合蛋白,并纯化该片段作为抗原免疫家兔,再以CNBr活化Sepharose4B微珠纯化抗血清制备PHF8特异性多克隆抗体。Western blotting以及免疫荧光检测表明该抗体具有很好的特异性,同时免疫荧光染色的结果也表明PHF8蛋白定位于细胞核。     

Passive Immunization with Phospho-Tau Antibodies Reduces Tau Pathology and Functional Deficits in Two Distinct Mouse Tauopathy Models

In Alzheimer’s disease (AD), an extensive accumulation of extracellular amyloid plaques and intraneuronal tau tangles, along with neuronal loss, is evident in distinct brain regions. Staging of tau pathology by postmortem analysis of AD subjects suggests a sequence of initiation and subsequent spread of neurofibrillary tau tangles along defined brain anatomical pathways. Further, the severity of cognitive deficits correlates with the degree and extent of tau pathology. In this study, we demonstrate that phospho-tau (p-tau) antibodies, PHF6 and PHF13, can prevent the induction of tau pathology in primary neuron cultures. The impact of passive immunotherapy on the formation and spread of tau pathology, as well as functional deficits, was subsequently evaluated with these antibodies in two distinct transgenic mouse tauopathy models. The rTg4510 transgenic mouse is characterized by inducible over-expression of P301L mutant tau, and exhibits robust age-dependent brain tau pathology. Systemic treatment with PHF6 and PHF13 from 3 to 6 months of age led to a significant decline in brain and CSF p-tau levels. In a second model, injection of preformed tau fibrils (PFFs) comprised of recombinant tau protein encompassing the microtubule-repeat domains into the cortex and hippocampus of young P301S mutant tau over-expressing mice (PS19) led to robust tau pathology on the ipsilateral side with evidence of spread to distant sites, including the contralateral hippocampus and bilateral entorhinal cortex 4 weeks post-injection. Systemic treatment with PHF13 led to a significant decline in the spread of tau pathology in this model. The reduction in tau species after p-tau antibody treatment was associated with an improvement in novel-object recognition memory test in both models. These studies provide evidence supporting the use of tau immunotherapy as a potential treatment option for AD and other tauopathies.     

Human fetal tau protein isoform: possibilities for Alzheimer's disease treatment

While early 1990s reports showed the phosphorylation pattern of fetal tau protein to be similar to that of tau in paired helical filaments (PHF) in Alzheimer's disease (AD), neither the molecular mechanisms of the transient developmental hyperphosphorylation of tau nor reactivation of the fetal plasticity due to re-expression of fetal protein kinases in the aging and AD human brain have been sufficiently investigated. Here, we summarize the current knowledge on fetal tau, adding new data on the specific patterns of tau protein and mRNA expression in the developing human brain as well as on change in tau phosphorylation in the perforant pathway after entorhinal cortex lesion in mice. As fetal tau isoform does not form PHF even in a highly phosphorylated state, understanding its expression and post-translational modifications represents an important avenue for future research towards the development of AD treatment and prevention.     

Phosphorylated tau protein is integrated into paired helical filaments in Alzheimer's disease

Antisera to paired helical filaments (PHF) were found to contain a significant amount of tau antibodies specific for a phosphorylated form, but only a negligible amount of those specific for a non-phosphorylated form. Also, the phosphorylated tau-specific antibodies, but not the non-phosphorylated tau-specific ones, labeled neurofibrillary tangles isolated in the presence of sodium dodecyl sulfate (SDS) and stained both tangles and senile plaque neuritis in fixed tissue sections in a very similar way to as the whole antiserum did. Taken together, these results strongly suggest that a major antigenic determinant of PHF is phosphorylated tau itself.     

PHF10 isoforms are phosphorylated in the PBAF mammalian chromatin remodeling complex

Chromatin remodeling complex PBAF(SWI/SNF) alters the structure of chromatin and controls gene expression. PHF10 is a specific subunit of PBAF complex and is expressed as four isoforms in mammalian cells. We demonstrated that all isoforms are expressed in various human cell types of different histological origins. All four isoforms are extensively phosphorylated and their phosphorylation level is depended on the cell type. Phosphorylation of PHF10 isoforms occurs while they are incorporated as a subunit of the PBAF complex, and therefore phosphorylation of PHF10 isoforms may play an essential role in regulation of PBAF complex’s function and mechanism of action.     

Fetal-Type Phosphorylation of the τ in Paired Helical Filaments

To determine the phosphorylation sites of the tau in paired helical filaments (PHF), two types of PHF antisera with different specificities were used: One was a conventional anti-PHF, and the other was an antiserum to formic acid-denatured PHF (anti-HFoPHF). Phosphorylated tau-specific antibodies, anti-ptau 1 and anti-ptau 2, were prepared from anti-PHF and anti-HFoPHF, respectively. We found that both anti-ptau 1 and anti-ptau 2 labeled fetal or juvenile tau but not adult tau. The anti-ptau 1- and anti-ptau 2-recognition sites were immunochemically localized to the fragment Asp313 to Ile328 in the most COOH-terminal portion of tau. Furthermore, Ser315 was determined as the anti-ptau 2 recognition site. The sequence surrounding Ser315 was not found in the canonical sequences phosphorylated with known kinases.     

Improved detection of substantia nigra pathology in Alzheimer's disease.

The role of substantia nigra pathology in Alzheimer's disease (AD) is uncertain. Detection of pathology may be obscured by intraneuronal neuromelanin and influenced by stains. We determined methods for optimal visualization of nigral pathology in 45 cases of AD. For detection of Lewy bodies (LBs), we compared ubiquitin and alpha-synuclein immunostains to hematoxylin and eosin (H&E). For neurofibrillary tangles (NFTs) and neuropil threads (NTs), we compared Gallyas silver and paired helical filament (PHF) immunostains, after bleaching of melanin, to modified Bielschowsky, Gallyas, and PHF alone. The number of LB cases was not different using the three stains. However, more LBs per section were detected using alpha-synuclein (z=4.88, p<0.001). Twice the number of cases exhibited NFT (z=8.21; p<0.001) and the mean NFT number per section was 2.8-5.2-fold greater, using Gallyas and PHF after bleaching compared to without bleaching (chi(2)=142.17; p<0.001). More NTs (z=6.54; p<0.001) were observed with PHF and Gallyas after bleaching. With optimal methods, we found LBs in 27%, NFTs in 89%, and NTs in all 45 AD cases. We show that detection of nigra pathology is influenced by histological method. Clinicopathological studies using these methods are needed to determine the role of nigral pathology in AD.     

Carboxyl-terminal fragments of presenilin-1 are closely related to cytoskeletal abnormalities in Alzheimer's brains

To clarify the role of presenilin-1 (PS-1) in the pathology of Alzheimer's disease (AD), we tested four antisera to PS-1. The specific antisera to the N-terminus (HSN-2) and C-terminus (HS-C) of PS-1 detected a 44/40kD holoprotein, a 25kD N-terminal fragment (NTF) and a 16kD C-terminal fragment (CTF) of PS-1 in COS-7 cells. The 25kD NTF and 16kD CTF were observed in human brains, and their amounts were not significantly different between the control and AD brains. The antibody HS-C labeled extensive neurofibrillary tangles, dystrophic neurites and curly fibers in the AD brains. In the paired helical filament (PHF) fraction containing A68 protein from AD brains, a smear pattern of CTFs was revealed. Antisera (HS-L292 and HS-L300) to cleavage sites of PS-1 also revealed immunoreactive neurofibrillary tangles in the AD brain sections and the smear pattern of CTFs of A68 protein fraction. The CTFs of PS-1 accumulate with PHF tau, suggesting a close relationship between PS-1 and cytoskeletal abnormalities in AD brains.