Formation Of Neurofibrillary Lesions Research Articles

Effects of anti-tau immunotherapy on reactive microgliosis, cerebral endotheliopathy, and cognitive function in an experimental model of cerebral malaria.

Cerebral malaria (CM), a potentially fatal encephalopathy caused primarily by infection with Plasmodium falciparum, results in long-term adverse neuro-psychiatric sequelae. Neural cell injury contributes to the neurological deficits observed in CM. Abnormal regulation of tau, an axonal protein pathologically associated with the formation of neurofibrillary lesions in neurodegenerative diseases, has been linked to inflammation and cerebral microvascular compromise and has been reported in human and experimental CM (ECM). Immunotherapy with a monoclonal antibody to pathological tau (PHF-1 mAB) in experimental models of neurodegenerative diseases has been reported to mitigate cognitive decline. We investigated whether immunotherapy with PHF-1 mAB prevented cerebral endotheliopathy, neural cell injury, and neuroinflammation during ECM. Using C57BL/6 mice infected with either Plasmodium berghei ANKA (PbA), which causes ECM, Plasmodium berghei NK65 (PbN), which causes severe malaria, but not ECM, or uninfected mice (Un), we demonstrated that when compared to PbN infection or uninfected mice, PbA infection resulted in significant memory impairment at 6 days post-infection, in association with abnormal tau phosphorylation at Ser202 /Thr205 (pSer202 /Thr205 ) and Ser396-404 (pSer396-404 ) in mouse brains. ECM also resulted in significantly higher expression of inflammatory markers, in microvascular congestion, and glial cell activation. Treatment with PHF-1 mAB prevented PbA-induced cognitive impairment and was associated with significantly less vascular congestion, neuroinflammation, and neural cell activation in mice with ECM. These findings suggest that abnormal regulation of tau protein contributes to cerebral vasculopathy and is critical in the pathogenesis of neural cell injury during CM. Tau-targeted therapies may ameliorate the neural cell damage and subsequent neurocognitive impairment that occur during disease.

Mass Spectrometry Analysis of Lysine Posttranslational Modifications of Tau Protein from Alzheimer's Disease Brain.

Recent advances in mass spectrometry (MS)-based proteomics have greatly facilitated the robust identification and quantification of posttranslational modifications (PTMs), including those that are present at substoichiometric site occupancies. The abnormal posttranslational modification and accumulation of the microtubule-associated protein tau has been implicated in the pathogenesis of Alzheimer's disease (AD), and it is thought that the primary mode of regulation of tau occurs through PTMs. Several studies have been published regarding tau phosphorylation; however, other tau PTMs such as ubiquitylation, acetylation, methylation, oxidation, sumoylation, nitration, and glycosylation have not been analyzed as extensively. The comprehensive detection and delineation of these PTMs is critical for drug target discovery and validation. Lysine-directed PTMs including ubiquitylation, acetylation, and methylation play key regulatory roles with respect to the rates of tau turnover and aggregation. MS-based analytical approaches have been used to gain insight into the tau lysine-directed PTM signature that is most closely associated with neurofibrillary lesion formation. This chapter provides details pertaining to the liquid chromatography tandem mass spectrometry (LC-MS/MS)-based analysis of the lysine-directed posttranslational modification of tau.

Neuroprotective Mechanisms Mediated by CDK5 Inhibition.

Cyclin-dependent kinase 5 (CDK5) is a proline-directed serine/threonine kinase belonging to the family of cyclin-dependent kinases. In addition to maintaining the neuronal architecture, CDK5 plays an important role in the regulation of synaptic plasticity, neurotransmitter release, neuron migration and neurite outgrowth. Although various reports have shown links between neurodegeneration and deregulation of cyclin-dependent kinases, the specific role of CDK5 inhibition in causing neuroprotection in cases of neuronal insult or in neurodegenerative diseases is not wellunderstood. This article discusses current evidence for the involvement of CDK5 deregulation in neurodegenerative disorders and neurodegeneration associated with stroke through various mechanisms. These include upregulation of cyclin D1 and overactivation of CDK5 mediated neuronal cell death pathways, aberrant hyperphosphorylation of human tau proteins and/or neurofilament proteins, formation of neurofibrillary lesions, excitotoxicity, cytoskeletal disruption, motor neuron death (due to abnormally high levels of CDK5/p25) and colchicine- induced apoptosis in cerebellar granule neurons. A better understanding of the role of CDK5 inhibition in neuroprotective mechanisms will help scientists and researchers to develop selective, safe and efficacious pharmacological inhibitors of CDK5 for therapeutic use against human neurodegenerative disorders, such as Alzheimer's disease, amyotrophic lateral sclerosis and neuronal loss associated with stroke.

Structure and mechanism of action of tau aggregation inhibitors.

Since the discovery of phenothiazines as tau protein aggregation inhibitors, many additional small molecule inhibitors of diverse chemotype have been discovered and characterized in biological model systems. Although direct inhibition of tau aggregation has shown promise as a potential treatment strategy for depressing neurofibrillary lesion formation in Alzheimer's disease, the mechanism of action of these compounds has been unclear. However, recent studies have found that tau aggregation antagonists exert their effects through both covalent and non-covalent means, and have identified associated potency and selectivity driving features. Here we review small-molecule tau aggregation inhibitors with a focus on compound structure and inhibitory mechanism. The elucidation of inhibitory mechanism has implications for maximizing on-target efficacy while minimizing off-target side effects.

Dual modification of Alzheimer’s disease PHF-tau protein by lysine methylation and ubiquitylation: a mass spectrometry approach

In sporadic Alzheimer’s disease (AD), neurofibrillary lesion formation is preceded by extensive post-translational modification of the microtubule associated protein tau. To identify the modification signature associated with tau lesion formation at single amino acid resolution, immunopurified paired helical filaments were isolated from AD brain and subjected to nanoflow liquid chromatography–tandem mass spectrometry analysis. The resulting spectra identified monomethylation of lysine residues as a new tau modification. The methyl-lysine was distributed among seven residues located in the projection and microtubule binding repeat regions of tau protein, with one site, K254, being a substrate for a competing lysine modification, ubiquitylation. To characterize methyl lysine content in intact tissue, hippocampal sections prepared from post mortem late-stage AD cases were subjected to double-label confocal fluorescence microscopy using anti-tau and anti-methyl lysine antibodies. Anti-methyl lysine immunoreactivity colocalized with 78 ± 13% of neurofibrillary tangles in these specimens. Together these data provide the first evidence that tau in neurofibrillary lesions is post-translationally modified by lysine methylation.Electronic supplementary materialThe online version of this article (doi:10.1007/s00401-011-0893-0) contains supplementary material, which is available to authorized users.

Pseudophosphorylation of tau protein directly modulates its aggregation kinetics

Hyperphosphorylation of tau protein is associated with neurofibrillary lesion formation in Alzheimer's disease and other tauopathic neurodegenerative diseases. It fosters lesion formation by increasing the concentration of free tau available for aggregation and by directly modulating the tau aggregation reaction. To clarify how negative charge incorporation into tau directly affects aggregation behavior, the fibrillization of pseudophosphorylation mutant T212E prepared in a full-length four-repeat tau background was examined in vitro as a function of time and submicromolar tau concentrations using electron microscopy assay methods. Kinetic constants for nucleation and extension phases of aggregation were then estimated by direct measurement and mathematical simulation. Kinetic analysis revealed that pseudophosphorylation increased tau aggregation rate by increasing the rate of filament nucleation. In addition, it increased aggregation propensity by stabilizing mature filaments against disaggregation. The data suggest that incorporation of negative charge into the T212 site can directly promote tau filament formation at multiple steps in the aggregation pathway.

Chronic oxidative stress causes increased tau phosphorylation in M17 neuroblastoma cells

Tau hyperphosphorylation appears to be a critical event leading to abnormal aggregation and disrupted function of tau in affected neurons in Alzheimer's disease (AD). As a prominent early event during AD pathogenesis, oxidative stress is believed to contribute to tau phosphorylation and the formation of neurofibrillary lesions. However, acute oxidative stress has disparate effects on tau phosphorylation. Given the chronic nature of AD, in this study, we aimed to determine the long-term effect of oxidative stress on tau phosphorylation. In this regard, we established a novel in vitro model of chronic oxidative stress through inhibition of glutathione (GSH) synthesis with BSO. We confirmed that these cells were under a chronic mild oxidative stress by looking at oxidative response, the induction of heme oxygenase 1 (HO-1) without neuronal death. Chronic oxidative stress increased levels of tau phosphorylated at PHF-1 epitope (serine 399/404) in a time-dependent manner. Our data further suggest that increased activity of JNK and p38 and decreased activity of PP2A are likely involved in chronic oxidative stress-induced tau phosphorylation. In conclusion we suggest that chronic oxidative stress contributes to increased tau phosphorylation in vitro and could play a critical role in neurofibrillary pathology in vivo.

Small-Molecule Mediated Neuroprotection in an In Situ Model of Tauopathy

Small-molecule inhibitors of neurofibrillary lesion formation may have utility for treatment of Alzheimer's disease and certain forms of frontotemporal lobar degeneration. These lesions are composed largely of tau protein, which aggregates to form intracellular fibrils in affected neurons. Previously it was shown that chronic overexpression of human tau protein within identified neurons (anterior bulbar cells) of the sea lamprey induced a phenotype-resembling tauopathic neurodegeneration, including the formation of tau filaments, fragmentation of dendritic arbors, and eventual cell death. Development of this neurodegenerative phenotype was blocked by chronic administration of a benzothiazole derivative termed N3 ((E)-2-[[4-(dimethylamino)phenyl]azo]-6-methoxybenzothiazole) to lamprey aquaria. Here we examined the mechanism of action of N3 and an alkene analog termed N4 ((E)-2-[2-[4-(dimethylamino)phenyl]ethenyl]-6-methoxybenzothiazole) in vitro and in the lamprey model. Results showed that although both compounds entered the lamprey central nervous system, only N3 arrested tauopathy. On the basis of in vitro aggregation assays, neither compound was capable of directly inhibiting tau filament formation. However, N3, but not N4, was capable of partially antagonizing the binding of Thioflavin S to synthetic tau filaments. The results suggest that occupancy of N3-binding sites on nascent tau filaments may significantly retard the progressive degeneration accompanying tau overexpression in lamprey.

Pathogenic missense MAPT mutations differentially modulate tau aggregation propensity at nucleation and extension steps

Mutations in the MAPT gene encoding tau protein lead to neurofibrillary lesion formation, neurodegeneration, and cognitive decline associated with frontotemporal lobar degeneration. While some pathogenic mutations affect MAPT introns, resulting in abnormal splicing patterns, the majority occur in the tau coding sequence leading to single amino acid changes in tau primary structure. Depending on their location within the polypeptide chain, tau missense mutations have been reported to augment aggregation propensity. To determine the mechanisms underlying mutation-associated changes in aggregation behavior, the fibrillization of recombinant pathogenic mutants R5L, G272V, P301L, V337M, and R406W prepared in a full-length four-repeat human tau background was examined in vitro as a function of time and submicromolar tau concentrations using electron microscopy assay methods. Kinetic constants for nucleation and extension phases of aggregation were then estimated by direct measurement and mathematical simulation. Results indicated that the mutants differ from each other and from wild-type tau in their aggregation propensity. G272V and P301L mutations increased the rates of both filament nucleation and extension reactions, whereas R5L and V337M increased only the nucleation phase. R406W did not differ from wild-type in any kinetic parameter. The results show that missense mutations can directly promote tau filament formation at different stages of the aggregation pathway.

O2-05-07: How do Tau isoform expression patterns influence neurofibrillary lesion formation in tauopathies?

O2-05-07: How do Tau isoform expression patterns influence neurofibrillary lesion formation in tauopathies?

Detection and quantification of tau aggregation using a membrane filter assay

Aggregation of the microtubule-associated protein tau contributes to the formation of neurofibrillary lesions in Alzheimer’s disease and is a useful marker of disease progression. Although filter trap assays have been employed to assess the extent of tau aggregation in cells and tissues as well as in vitro, their performance relative to other assay modalities has not been reported. To clarify this issue, the ability of the filter trap approach to quantify aggregation of purified recombinant full-length tau protein in vitro was examined as a function of membrane chemistry in a 96-well format. Results showed that nitrocellulose yielded the greatest assay sensitivity relative to polyvinylidene fluoride or cellulose acetate at equal membrane porosity. However, all combinations of filter chemistries, porosities, and monoclonal detection antibodies yielded nonlinear correlations between signal intensity and analyte concentration. When corrected for nonlinearity, the filter trap assay determined a value for the critical monomer concentration for tau aggregation that was statistically identical to determinations made by electron microscopy assay. The data suggest conditions under which filter trap assays can be used to estimate tau aggregation kinetics.

C-terminal truncation modulates both nucleation and extension phases of τ fibrillization

Proteolytic post-translational modification has been proposed as an early stage event in the aggregation of τ protein and formation of neurofibrillary lesions in Alzheimer’s disease. Caspases and other proteases cleave τ in vivo at discrete locations including Asp 421 and Glu 391. Both cleavage products are prone to aggregation relative to wild-type, full-length τ protein. To determine the mechanism underlying this effect, the fibrillization of τ truncated after Asp 421 and Glu 391 residues was characterized in a full-length four-repeat τ background using quantitative electron microscopy methods under homogeneous nucleation conditions. Both C-terminal truncations decreased critical concentration relative to full-length τ, resulting in more filament mass at reaction plateau. Moreover, truncation directly augmented the efficiency of the nucleation reaction. The results suggest the mechanism through which C-terminal proteolysis can modulate τ filament accumulation depending on whether it precedes or follows nucleation.

Elevated levels of tau in cerebrospinal fluid: implications for the antemortem diagnosis of Alzheimer's disease elevated levels of tau in cerebrospinal fluid: implications for the antemortem diagnosis of Alzheimer's disease.

Alzheimer's disease (AD) is a heterogeneous group of dementias characterized by progressive cognitive impairments as well as by the accumulation of abundant extracellular deposits of Ass and intra-neuronal neurofibrillary lesions in selective-ly vulnerable regions of the AD brain. The latter abnormalities (e.g. neurofibrillary tangles, dystrophic neurites, neuropil threads) are aggregates of paired helical filaments (PHFs) formed from altered tau proteins (PHFtau). Although PHF tau and normal central nervous system (CNS) tau are phosphorylated at nearly the same sites, PHFtau is phosphorylated to a greater extent, and alterations in the activity of CNS kinases and phosphatases most likely contribute to the pathogenesis of PHFtau. Since the abundance of neurofibrillary lesions correlates with the dementia in AD, the generation of PHFtau and the formation of neurofibrillary lesions may be part of a cell death pathway leading to massive neuron loss and dementia in AD. Building upon these and other insights into altered tau metabolism in AD, a series of studies suggest that the diagnosis of AD may be supported in living patients by determining the concentration of tau in cerebrospinal fluid (CSF). We review these promising studies here, and discuss them in the context of current understanding of the pathobiology of AD.

Phosphorylation Activity in the Alzheimer's Disease and Normal Brain is Modulated by Microtubule-Associated Protein, Tau in Vitro.

One of the hallmarks of Alzheimer's Disease is the presence of abundant neurofibrillary tangles (NFTs) in the brains of affected individuals. Hyperphosphorylated tau is a major component of paired helical filaments (PHFs) in NFTs. Tau is a neuronal microtubule associated protein found primarily in axons. Normal tau promotes tubulin polymerization and stabilizes microtubule (MT) structures, whereas hyperphosphorylated tau reduces its affinity for MTs and destabilizes MT-structures. This results in the disruption of vital cellular processes (e.g. axonal transport) and leads to the degeneration of affected neurons. Processes leading to the hyperphosphorylation of tau and formation of neurofibrillary lesions in Alzheimer's Disease (AD) brains are not understood. Phosphorylation of a substrate molecule like tau depends upon the equilibrium between kinase and phosphatase activities and the availability of their substrate molecules in a given system. Therefore, to understand the relative roles of kinase and phosphatase activities, we studied the long-term kinetics of phosphorylation in AD and control brain extracts in the presence and absence of the phosphatase inhibitor okadaic acid (OA) using histone, casein and bacterially expressed tau as exogenous substrates. It was found that both kinase and phosphatase activities were higher in AD compared to control brains. Surprisingly, between 18 and 24 hours, there was a robust increase in phosphorylation of endogenous proteins in the brain extracts only when bacterially expressed tau was present in the phosphorylation reaction mixture. This pattern of phosphorylation activity was unaffected by OA. Significant difference in the phosphorylation of tau isoforms was also seen during this period. These data suggest that the expression and differential phosphorylation of certain tau isoforms may be responsible for the robust increase in phosphorylation and may play an important role in Alzheimer's pathology.

Neuropathology of Alzheimer’s disease

In the present review we describe the morphological features of Alzheimer's disease (AD) and compare these findings with those obtained in argyrophilic grain disease, a frequent but often unrecognized form of late-onset dementia. Macroscopically AD brains exhibit a marked atrophy of the medical temporal lobe, including the hippocampus, entorhinal cortex and amygdala. Neuronal loss, decreased synapse density and the intra- and extracellular deposition of abnormal proteins constitute the histological hallmark lesions of AD. The intraneural accumulation of the microtubule-associated protein tau in a hyperphosphorylated state leads to the formation of neurofibrillary lesions (NFL). Whereas the widespread distribution of NFL in the neocortex correlates with the cognitive decline in AD patients no such correlation could be found for the extracellular deposition of the A beta-protein in the shape of senile plaques (SP). However, dementia correlates with the amount of neuritic degeneration within a subtype of SP ('neuritic plaques'). We further discuss some of the risk factors for AD, i.e. the genetic risk factors.

Chapter 21 Neurofibrillary pathology of Alzheimer's disease and other tauopathies

Publisher Summary Alzheimer's disease is the most common neurodegenerative disorder. It affects 15-20 million people worldwide and constitutes the fourth leading cause of death in the industrialized world. Its major symptoms are a progressive loss of memory and other cognitive functions, resulting in a severe and irreversible dementia. The intellectual decline is accompanied by the accumulation in the brain of insoluble fibrous material, extracellularly in the form of plaques composed of amyloid protein AP, and intracellularly in the form of neurofibrillary lesions. A study of the mechanisms that lead to the formation of plaques and neurofibrillary lesions is essential for an understanding of the pathogenesis of all forms of Alzheimer's disease. The extent and topographical distribution of neurofibrillary lesions provide a reliable pathological correlate of the degree of dementia. The formation of neurofibrillary lesions is therefore believed to lead to the symptoms of Alzheimer's disease, which result most probably from the degeneration of nerve cells in cerebral cortex and hippocampal formation, with ensuing neuronal cell loss and reduction in synapse number. The finding of large numbers of amyloid deposits in some cognitively normal individuals (also known as “pathological ageing”) has shown that amyloid deposits are not sufficient for dementia. In contrast, large numbers of neurofibrillary lesions in hippocampus and/or cerebral cortex are always accompanied by dementia. Over recent years, it has become apparent that in other forms of dementia filamentous tau deposits are frequently observed in the absence of amyloid deposits. This chapter reviews the tau pathology of Alzheimer's disease and other “tauopathies.”

Interaction of aluminum with paired helical filament tau is involved in neurofibrillary pathology of Alzheimer's disease.

Since the first reports of aluminum-induced neurofibrillary degeneration in experimental animals, extensive studies have been performed to clarify the role played by aluminum in the pathogenesis of Alzheimer's disease (AD). Additional evidence implicating aluminum in AD includes elevated levels of aluminum in the AD brain, epidemiologic data linking aluminum exposure to AD, and interactions between aluminum and protein components in the pathologic lesions of AD, i.e., neurofibrillary tangles (NFTs) and senile plaques. As most of this evidence is circumstantial and some of it is not consistent in all reports, the role of aluminum in the pathogenesis of AD has remained controversial. However, the interaction of aluminum with altered forms of tau in the paired helical filaments (PHFs) of neurofibrillary lesions is highly likely to contribute to the formation of NFTs because (1) aluminum and abnormally phosphorylated tau (known as PHF tau) are colocalized in NFTs, and (2) aluminum is known to preferentially interact with such phosphorylated proteins. Recently, it was demonstrated that aluminum binds selectively to PHF tau, induces PHF tau to aggregate, and retards the in vivo proteolysis of PHF tau. These data suggest that aluminum could serve as a cofactor in the formation of NFTs by interacting with PHF tau. This review summarizes the current understanding of how aluminum might contribute to the formation of neurofibrillary lesions from PHF tau in neurons of the AD brain.

Regulation of tau phosphorylation in Alzheimer's disease.

Alzheimer's disease (AD) is a heterogeneous dementing disorder of the elderly that is characterized by progressive cognitive impairments and the accumulation of abundant amyloid or senile plaques (SPs) and neurofibrillary tangles (NFTs) as well as the massive loss of neurons in the AD brain. Indeed, a secure diagnosis of AD in patients with a chronic progressive dementia requires evidence of numerous SPs and NFTs in the postmortem brain. Although the deposition of fibrillar amyloid or A beta-peptides in extracellular plaques and the accumulation of tau-rich intraneuronal NFTs are not restricted exclusively to AD, there is a close correlation between the burden of tau-rich neurofibrillary lesions in selected telencephalic regions of the brain and the dementia in AD. Since the formation of neurofibrillary lesions from hyperphosphorylated tau proteins may compromise the function and viability of neurons in the AD brain, this review summarizes recent insights into mechanisms that regulate the phosphorylation state of tau in AD.