Formation Of Paired Helical Filaments Research Articles

Strategies to delay the onset of Alzheimer's disease.

Several processes are implicated in the neuropathology of Alzheimer's disease (AD), such as the deposition of amyloid, the formation of paired helical filaments and the proinflammatory activation of microglial and astroglial cells. Proinflammatory activation of glial cells has been a focus of research for a mere ten years now. However, the availability of and broad experience with anti-inflammatory drugs has led to several ongoing clinical trials to verify the capacity of anti-inflammatory drugs to ameliorate the deterioration in AD. The enzymatic cleavage of the amyloid-precursor-protein or the hyperphosphorylation of tau as well as the subsequent aggregation of the resulting products are further targets for drugs intended to delay the neuropathological destruction observed in AD.

Fibers of tau fragments, but not full length tau, exhibit a cross beta-structure: implications for the formation of paired helical filaments.

We have used X-ray fiber diffraction to probe the structure of fibers of tau and tau fragments. Fibers of fragments from the microtubule binding domain had a cross beta-structure that closely resembles that reported both for neurofibrillary tangles found in Alzheimer's disease brain and for fibrous lesions from other protein folding diseases. In contrast, fibers of full-length tau had a different, more complex structure. Despite major differences at the molecular level, all fiber types exhibited very similar morphology by electron microscopy. These results have a number of implications for understanding the etiology of Alzheimer's and other tauopathic diseases. The morphology of the peptide fibers suggests that the region in tau corresponding to the peptides plays a critical role in the nucleation of fiber assembly. The dramatically different structure of the full length tau fibers suggests that some region in tau has enough inherent structure to interfere with the formation of cross beta-fibers. Additionally, the similar appearance by electron microscopy of fibrils with varying molecular structure suggests that different molecular arrangements may exist in other samples of fibers formed from tau.

Induction of Alzheimer-specific Tau epitope AT100 in apoptotic human fetal astrocytes.

In Alzheimer's and other neurodegenerative diseases, hyperphosphorylated tau accumulates in affected neuronal and glial cells in the form of paired helical filaments (PHFs). This tau binds antibody AT100, which recognizes the double phosphorylation site (Thr212/Ser214) that is not present in normal biopsy tau. In primary cultures, highly enriched (>98%) in astrocytes of human fetal brain, three polypeptides of 52, 64, and 70 kD showed immunoreactivity with tau antibodies against non-phosphorylated epitopes, accounting for 88, 12, and <1%, respectively, of the total reactivity. All three polypeptides were phosphorylated at the PHF-1 epitope but not at the epitopes Tau-1, 12E8, AT8, and AT100. Treatment of cultures with okadaic acid resulted in apoptosis characterized by the blebbing of the plasma membrane, condensation of nuclear chromatin, and fragmentation of the nucleus. This treatment also resulted in a 3- to 5-fold increase in the content of both tau protein and phosphorylation. The increases were observed in all phosphorylation sites examined, and included the AT100 site. The AT100 site has been proposed to be generated by protein kinase B/Akt and Cdc2. Since okadaic acid can induce an AD-like hyperphosphorylated state of normal tau in primary cultures of human brain cells, a simple cellular model is available permitting study of self-aggregation of tau and phosphorylation events characteristic of neurodegeneration.

Formation of diffuse and fibrillar tangles in aging and early Alzheimer’s disease ☆

The changes in tau that are associated with the early formation of tangles in aging and in preclinical and very mild Alzheimer’s Disease (AD) were studied with two antibodies against AD-specific tau: PHF-1, which recognizes a phosphorylated epitope at Ser396 through 404, and MC-1, which recognizes a folded, conformational epitope that includes amino acids at both 7 through 9 and 312 through 342. Both antibodies demonstrated cells with diffuse or granular staining (diffuse tangles) and cells with fibrillar staining (fibrillar tangles). The fibrillar tangles corresponded to classical tangles and increase exponentially with age and severity of AD. The diffuse tangles seemed to represent an earlier form of tangles; their density peaked around preclinical AD, and then decreased in more severe stages of AD. MC-1 consistently stained more diffuse tangles than PHF-1, suggesting that the conformational change in tau precedes phosphorylation at the PHF-1 epitope during paired helical filament formation.

Cdk5 and munc-18/p67 co-localization in early stage neurofibrillary tangles-bearing neurons in Alzheimer type dementia brains.

Hyperphosphorylation of tau protein occurs during the formation of paired helical filament (PHF) in the brain with Alzheimer's disease. As previously reported, cyclin-dependent kinase (cdk) 5 can phosphorylate tau at the site of abnormally phosphorylated in PHF. To characterize the relationship between cdk5 and PHF-tau, we investigated the localization of cdk5 and its regulator, p67 (munc 18), in the hippocampus and temporal lobes from 12 Alzheimer type dementia (ATD) patients and 5 controls using immunohistochemical procedures. The specificity of antibodies was confirmed with Western blot analysis. Anti-cdk5 antibody diffusely stained the perikarya of some tau2-positive or neurofibrillary tangle (NFT)-bearing neurons in ATD brains, while cdk5-positive staining was scarcely found in control brains. Anti-p67 antibody also showed stronger immunoreactivity of pyramidal neurons in ATD brains than in control brains. Double immunostaining with anti-cdk5 and anti-p67 antibodies revealed co-localization of both molecules in some pyramidal neurons. These findings suggest that cdk5 is activated by p67 at the early stage of NFT formation and accelerates NFT formation. In cdk5-positive and p67-negative neurons, cdk5 may be activated by other regulator molecules such as p35. In addition, cdk5-positive reactive astrocytes were found close to cdk5-positive NFT-bearing neurons m ATD brains but not in control brains, suggesting a correlation between NFT and reactive astrocytes.

Elevated transglutaminase-induced bonds in PHF tau in Alzheimer's disease

Transglutaminase-induced ε-(γ-glutamyl)lysine bonds covalently cross-link and polymerize peptides into insoluble high molecular weight protein aggregates resistant to degradation and proteolytic digestion. We investigated the hypothesis that excessive deposition of ε-(γ-glutamyl)lysine bonds is a neuropathological mechanism which induces the polymerization of tau protein into stable aggregates leading to the formation of paired helical filaments (PHFs) which deposit into neurofibrillary tangles in Alzheimer's disease (AD) brain. We demonstrate a significant (45%) elevation in ε-(γ-glutamyl)lysine cross-links in AD cortex as compared to control cortex. In vivo, PHF tau, and high and medium molecular weight neurofilament proteins have significantly greater cross-linking by ε-(γ-glutamyl)lysine bonds in AD brains as compared to controls. The cross-linking of PHF tau occurs both intra-molecularly and inter-molecularly. The inter-molecular cross-linking of tau could account for the formation of high molecular weight tau polymers. These results suggest that transglutaminase-induced cross-linking of tau protein could play a role in the formation and stabilization of neurofibrillary tangles. Inhibition of transglutaminase-induced cross-linking may therefore, provide a novel strategy for the treatment of AD.

Soluble Amyloid β Peptide Concentration as a Predictor of Synaptic Change in Alzheimer's Disease

We have characterized amyloid beta peptide (Abeta) concentration, Abeta deposition, paired helical filament formation, cerebrovascular amyloid angiopathy, apolipoprotein E (ApoE) allotype, and synaptophysin concentration in entorhinal cortex and superior frontal gyrus of normal elderly control (ND) patients, Alzheimer's disease (AD) patients, and high pathology control (HPC) patients who meet pathological criteria for AD but show no synapse loss or overt antemortem symptoms of dementia. The measures of Abeta deposition, Abeta-immunoreactive plaques with and without cores, thioflavin histofluorescent plaques, and concentrations of insoluble Abeta, failed to distinguish HPC from AD patients and were poor correlates of synaptic change. By contrast, concentrations of soluble Abeta clearly distinguished HPC from AD patients and were a strong inverse correlate of synapse loss. Further investigation revealed that Abeta40, whether in soluble or insoluble form, was a particularly useful measure for classifying ND, HPC, and AD patients compared with Abeta42. Abeta40 is known to be elevated in cerebrovascular amyloid deposits, and Abeta40 (but not Abeta42) levels, cerebrovascular amyloid angiopathy, and ApoE4 allele frequency were all highly correlated with each other. Although paired helical filaments in the form of neurofibrillary tangles or a penumbra of neurites surrounding amyloid cores also distinguished HPC from AD patients, they were less robust predictors of synapse change compared with soluble Abeta, particularly soluble Abeta40. Previous experiments attempting to relate Abeta deposition to the neurodegeneration that underlies AD dementia may have failed because they assayed the classical, visible forms of the molecule, insoluble neuropil plaques, rather than the soluble, unseen forms of the molecule.

Alterations in content and phosphorylation state of cytoskeletal proteins in the sciatic nerve during ageing and in Alzheimer's disease.

Paired helical filaments containing the microtubule-associated protein tau in an abnormally high phosphorylated state are one of the major hallmarks of Alzheimer's disease. In the central nervous system, this neurofibrillar degeneration preferentially affects long-axon projection neurons. In the peripheral nervous system largely made up by long-axon neurons, formation of paired helical filaments, however, has only rarely been described. In the present study, we have analysed alterations in the content and phosphorylation state of tau and neurofilament protein in the sciatic nerve during ageing and in Alzheimer's disease. The amount of both cytoskeletal proteins remained constant during ageing but was significantly reduced in Alzheimer's disease. The phosphorylation state of tau protein was elevated during ageing as well as in Alzheimer's disease. No indications of a paired helical filament-like aggregation of tau were found. It is concluded that during normal ageing and in Alzheimer's disease, processes are activated in the peripheral nervous system that induce a hyperphosphorylation of tau. Increased phosphorylation of tau in peripheral neurons, however, is not necessarily accompanied by the formation of paired helical filaments. Analysing principal differences in the expression, posttranslational modification and metabolism of tau between central and peripheral neurons might, therefore, help to get a better insight into the mechanism of paired helical filament formation.

The prolyl isomerase Pin1 restores the function of Alzheimer-associated phosphorylated tau protein.

One of the neuropathological hallmarks of Alzheimer's disease is the neurofibrillary tangle, which contains paired helical filaments (PHFs) composed of the microtubule-associated protein tau. Tau is hyperphosphorylated in PHFs, and phosphorylation of tau abolishes its ability to bind microtubules and promote microtubule assembly. Restoring the function of phosphorylated tau might prevent or reverse PHF formation in Alzheimer's disease. Phosphorylation on a serine or threonine that precedes proline (pS/T-P) alters the rate of prolyl isomerization and creates a binding site for the WW domain of the prolyl isomerase Pin1. Pin1 specifically isomerizes pS/T-P bonds and regulates the function of mitotic phosphoproteins. Here we show that Pin1 binds to only one pT-P motif in tau and copurifies with PHFs, resulting in depletion of soluble Pin1 in the brains of Alzheimer's disease patients. Pin1 can restore the ability of phosphorylated tau to bind microtubules and promote microtubule assembly in vitro. As depletion of Pin1 induces mitotic arrest and apoptotic cell death, sequestration of Pin1 into PHFs may contribute to neuronal death. These findings provide a new insight into the pathogenesis of Alzheimer's disease.

Elevated expression of p21 ras is an early event in Alzheimer's disease and precedes neurofibrillary degeneration

Alzheimer's disease is a chronic degenerative disorder characterized by the intracellular accumulation of “paired helical filaments” consisting of highly phosphorylated tau and by extracellular deposits of aggregated Aβ-peptide. Furthermore, neurodegeneration in Alzheimer's disease is associated with the appearance of neuritic growth profiles that are aberrant with respect to their localization, morphological appearance, and composition of cytoskeletal elements. 2 During early stages of Alzheimer's disease, a variety of growth factors and mitogenic compounds 10,12,15,16,18,32 are elevated. Most of these factors mediate their cellular effects through activation of the p21 ras-dependent mitogen-activated protein kinase cascade, a pathway that is also involved in the regulation of expression and post-translational modification of the amyloid precursor protein and tau protein. 7,11,13,14,19,20,22,31 We previously reported on the elevated expression of p21 ras associated with paired helical filament formation and Aβ-deposits. 17 However, the question arises as to whether induction of p21 ras and the downstream mitogen-activated protein kinase cascade is an early event with rather primary importance in the pathogenetic chain or simply occurs as a cellular response to neurodegeneration. 29 The present study shows that expression of p21 ras is clearly elevated in very early stages of the disease, preceding both neurofibrillary pathology and formation of Aβ.

Deamidation and Isoaspartate Formation in Smeared Tau in Paired Helical Filaments: UNUSUAL PROPERTIES OF THE MICROTUBULE-BINDING DOMAIN OF TAU

An extensive loss of a selected population of neurons in Alzheimer's disease is closely related to the formation of paired helical filaments (PHFs). The most striking characteristic of PHFs upon Western blotting is their smearing. According to a previously described protocol (Morishima-Kawashima, M., Hasegawa, M., Takio, K., Suzuki, M., Titani, K., and Ihara, Y. (1993) Neuron 10, 1151-1160), smeared tau was purified, and its peptide map was compared with that of soluble (normal) tau. A CNBr fragment from soluble tau (CN5; residues 251-419 according to the 441-residue isoform) containing the microtubule-binding domain migrated at 15 and 18 kDa on SDS-polyacrylamide gel electrophoresis, whereas that from smeared tau exhibited two larger, unusually broad bands at approximately 30 and approximately 45 kDa, presumably representing dimers and trimers of CN5. In the peptide map of smeared tau-derived CN5, distinct peaks eluting at unusual locations were noted. Amino acid sequence and mass spectrometric analyses revealed that these distinct peptides bear isoaspartate at Asn-381 and Asp-387. Because no unusual peptides other than aspartyl or isoaspartyl peptide were found in the digests of smeared tau-derived CN5, it is likely that site-specific deamidation and isoaspartate formation are involved in its dimerization and trimerization and thus in PHF formation in vivo.

Heparin injection into the adult rat hippocampus induces seizures in the absence of macroscopic abnormalities

The pathological hallmarks of Alzheimer's disease include neurofibrillary tangles, neuropil threads and neuritic plaques. Neurofibrillary tangles and neuropil threads are comprised of paired helical filaments which are themselves composed of a hyperphosphorylated form of the microtubule-associated protein tau. 10,13 Neuritic plaques are extracellular deposits of aggregated beta amyloid associated with neurites containing hyperphosphorylated tau. 3 The mechanisms by which the neurofibrillary tangles and neuritic plaques develop in Alzhemier's disease are not clear but it is hypothesized that sulphated glycosaminoglycans are important in their formation. This impression is based on the finding that the glycosaminoglycan, heparan sulphate, is found associated with neurofibrillary tangles, neuritic plaques and neuropil threads 8,16,18 while dermatan sulphate, chondroitin sulphate and keratan sulphate immunoreactivity is found around neuritic plaques 17 in brains of Alzheimer's disease patients. Furthermore, in vitro studies demonstrate that sulphated glycosaminoglycaus such as heparan sulphate and the closely related molecule heparin interact with tau and potentiate its phosphorylation by a number of serine/threonine kinases, reduce its ability to bind to microtubules and induce paired helical filament formation, all properties associated with tau isolated from Alzheimer's disease brain. 8,12 Thus, we were interested to learn whether intracerebral injection of the sulphated glycosaminoglycan heparin would give rise to alterations in the cytoskeletal protein tau in the rat brain. Although no cytoskeletal changes were observed, to our considerable surprise we found that the intrahippocampal injection of heparin gave rise to seizures. We have investigated this unexpected effect further in vivo and by using in vitro electrophysiological techniques.

The relevance of apolipoprotein E polymorphism to Alzheimer's disease

The apo E gene located on chromosome 19 in humans is polymorphic. The three apo E isoforms E2, E3, and E4 are coded by three common alleles of the gene. The amyloid plaques in brains of Alzheimer disease (AD) patients are known to contain apo E. There is an increased prevalence of E4 allele in AD patients. apo E exhibits increased binding to a peptide Aβ deriving from amylold precursor protein. apo E, the risk factor for late AD disease is unable to prevent formation of paired helical filaments which in turn destabilizes neuronal microtubules.A variety of molecular techniques are available for apo E genotyping using DNA amplified by the polymerase chain reaction (PCR). The high guanine to cytosine content of apo E is problematic to the extent that the yield of PCR product and hybridization stringency can be compromised. The specificity of diagnosis of late-onset AD can be improved when results of apo E genotyping are evaluated together with clinical criteria.

Age-related changes of DNA repair and mitochondrial DNA synthesis in the mouse brain

Using quantitative autoradiography, both nuclear DNA repair - measured as nuclear unscheduled DNA synthesis (UDS) - and mitochondrial (mt) DNA synthesis were evaluated in situ for several types of cells in the brains of untreated mice of various age. It was found that distinct types of neuronal cells showed a decline of both UDS and mtDNA synthesis with age, whereas - except for glial cells of the cerebral cortex - no glial or endothelial cells showed age-related alterations of UDS. Together with various data reported in the literature, these patterns of a cell type-specific decrease of UDS and mtDNA synthesis with age in the mouse brain lead to an improved understanding of the complex interrelationships between the molecular events associated with the phenomenon of aging as well as to a new idea regarding the cause of the specific distribution pattern of those cells in the human brain that are affected by the formation of paired helical filaments in Alzheimer's disease.

The Tauopathies: Toward an Experimental Animal Model

Abundant intraneuronal neurofibrillary lesions within certain brain regions constitute a defining neuropathological characteristic of Alzheimer’s disease. 1 Ultrastructurally, the neurofibrillary lesions consist of abnormal filamentous deposits in the form of paired helical filaments (PHFs) and the related straight filaments (SFs). These filaments are made of the microtubule-associated protein tau in a hyperphosphorylated state. In normal brain, tau protein is soluble and nonfilamentous. Its ordered assembly into filaments is therefore a pathological event. Tau pathology is not limited to Alzheimer’s disease but is also present in a number of other dementing disorders, such as Pick’s disease, progressive supranuclear palsy, and corticobasal degeneration. 2,3 In these disorders, as in Alzheimer’s disease, the hyperphosphorylated tau protein is filamentous. However, the filament morphologies and tau isoform compositions differ from those of Alzheimer’s disease. The good correlation between the presence of tau pathology and the degree of cognitive impairment has suggested that the events leading to the formation of tau filaments or the mere presence of these filaments are sufficient to produce nerve cell degeneration. Recently, this view has been significantly reinforced by the discovery of mutations in the tau gene in familial frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). 4-8 The new work will no doubt lead to increased efforts aimed at producing experimental animal models of the tau pathology of Alzheimer’s disease and other tauopathies.

Dendritic changes in Alzheimer's disease and factors that may underlie these changes

It seems likely that the Alzheimer disease (AD)-related dendritic changes addressed in this article are induced by two principally different processes. One process is linked to the plastic response associated with deafferentation, that is, long-lasting transneuronally induced regressive changes in dendritic geometry and structure. The other process is associated with severe alterations of the dendritic- and perikaryal cytoskeleton as seen in neurons with the neurofibrillary pathology of AD, that is, the formation of paired helical filaments formed by hyperphosphorylated microtubule-associated protein tau. As the development of dendritic and cytoskeletal abnormalities are at least mediated by alterations in signal transduction, this article also reviews changes in signal pathways in AD. We also discuss transgenic approaches developed to model and understand cytoskeletal abnormalities.

Alzheimer's disease as a defect of neuronal autotrophism: a hypothetical analogy with amateur radio operation

Amyloid precursor protein appears to be a signaling protein that plays a role in neuronal autotrophism, indicating integrity of the nerve terminal and synapse. Analogous to amateur radio operation with a damaged antenna in which further attempts to improve signal propagation can result in damage to the radio equipment, abnormal signaling by the secreted amyloid precursor protein stimulates compensatory metabolic activity in the neuron that ultimately leads to paired helical filament formation (neurofibrillary tangles) and further neuronal dysfunction and death.

Chloroquine myopathy suggests that tau is degraded in lysosomes: implication for the formation of paired helical filaments in Alzheimer's disease

We have found that amorphous tau deposits in chloroquine myopathy (CM), a vacuolar myopathy induced by the administration of chloroquine, a well-known lysosomotropic agent. The dynamics of tau in CM and immunocytochemistry strongly suggest that the accumulation of tau is due to defective tau degradation in the lysosomal compartment in the muscle. This observation may offer a new view on the formation of paired helical filaments in Alzheimer's disease: this selective protein degradation pathway may be defective and result in intracellular accumulation of tau, thereby forming the unusual filaments.

Reduction of O-LinkedN-Acetylglucosamine-Modified Assembly Protein-3 in Alzheimer’s Disease

Abnormal protein processing and modification is associated with Alzheimer's disease (AD) pathology. The role of phosphorylation in AD has been studied extensively because the presumed abnormal phosphorylation of tau protein is believed to play a role in the formation of paired helical filaments. Glycosylation with O-linked N-acetylglucosamine (O-GlcNAc) to serine and threonine residues is a dynamic protein modification of intracellular proteins, and it shares similar features with protein phosphorylation. In this study, O-GlcNAc glycosylation of proteins from autopsied human brains with confirmed AD and non-AD age-matched controls was examined. O-GlcNAcylation was demonstrated by labeling protein extracts with [3H]galactose in the presence of galactosyltransferase and subsequent analyses of saccharide-protein linkage and saccharide structure. The number of O-GlcNAc-containing proteins and the overall O-GlcNAc level do not appear to be different between AD and control brain tissues. The only significant change observed is a marked reduction of O-GlcNAcylated clathrin assembly protein-3 (AP-3) in AD. The reduction is more evident in brain neocortical regions, and there appears to be a negative correlation between O-glycosylated AP-3 and the density of neurofibrillary tangles. These data suggest a possible association between the O-glycosylated AP-3 and AD pathology.

Collapsin Response Mediator Protein-2 Is Associated with Neurofibrillary Tangles in Alzheimer's Disease

Intraneuronal accumulation of paired helical filaments (PHF) is considered to be closely related to the neuronal loss observed in brains of patients affected with Alzheimer's disease. The central issue is whether PHF formation itself causes or accelerates the neuronal perikaryal and neuritic degeneration or whether they are simply the consequence of preceding degeneration. We sought to address the issue in part by characterizing the PHF-associated molecules and thus raised a number of monoclonal antibodies to neurofibrillary tangles. One monoclonal antibody, 3F4, strongly reacted with neurofibrillary tangles and some plaque neurites but few neuropil threads. This monoclonal antibody labeled a 65-kDa protein, but not tau or ubiquitin, on a Western blot of human brain extract and immunoprecipitated the same protein. The peptides released from the purified 65-kDa protein had the same sequences as those of a newly identified protein, human collapsin response mediator protein-2. Incorporation into neurofibrillary tangles may deplete soluble, cytosolic human collapsin response mediator protein-2 and lead to abnormal neuritic and/or axonal outgrowth of the tangle-bearing neuron, thus accelerating the neuritic degeneration in Alzheimer's disease.