Extracellular Neurofibrillary Tangles Research Articles

Genetics and Molecular Biology of Alzheimer's Disease and Frontotemporal Lobar Degeneration

Alzheimer's disease (AD) is the most common cause of dementia in the elderly, whereas frontotemporal lobar degeneration (FTLD) is the most frequent neurodegenerative disorder with a pre-senile onset. The two major neuropathological hallmarks of AD are extracellular amyloid beta plaques and intracellular neurofibrillary tangles. In FTLD the deposition of tau has been observed in a number of cases, but in several brains there is no deposition of tau but instead a positivity for ubiquitin. In some families these diseases are inherited in an autosomal dominant fashion. Genes responsible for familial AD include the amyloid precursor protein (APP), presenilin 1 (PS1) and presenilin 2 (PS2), whereas the main genes responsible for familial FTLD are microtubule-associated protein tau gene (MAPT) and progranulin (GRN). Concerning sporadic AD, it is known that the presence of the ε4 allele of the apolipoprotein E gene is a susceptibility factor. A number of additional genetic factors contribute to susceptibility for AD and FTLD.

How do soluble oligomers of amyloid β-protein impair hippocampal synaptic plasticity?

How do soluble oligomers of amyloid β-protein impair hippocampal synaptic plasticity?

Mitochondrial failure in a transgenic mice model of Alzheimer's disease with plaques and tangles

Alzheimer's disease is characterized by neuropathological hallmarks of extracellular amyloid plaques and intracellular neurofibrillary tangles in the brain of patients. Amyloid plaques are composed of the amyloid-beta (Aβ) protein, derived from its precursor protein APP. Neurofibrillary lesions are formed from paired helical filaments composed of hyperphosphorylated tau protein. Importantly, current data indicate a complex relation between the amyloid pathology and pathology involving microtubule-associated protein tau during disease. Based on our previous findings, we hypothesize a direct impact of abnormally phosphorylated tau and Aβ on proteins/enzymes involved in energy metabolism and mitochondrial respiratory chain. For this approach we are currently investigating the brains of double (APP (KM670/671NL)/PS2(N141l)) and triple (APP(KM670/671NL)/PS2(N141l)/Tau(P301L)) transgenic mice at the age of 7–8, 12 and 16 months. Mitochondrial respiration is studied by the measurement of oxygen consumption with substrates and inhibitors specific for the mitochondrial chain complexes I and IV. In addition, enzyme activities of these complexes will be determined. Our preliminary findings indicate that at the level of mitochondria, the two defining neuropathological AD proteins, tau and A?, seem to act in a synergistic or additive way finally leading to/accelerating neurodegenerative mechanisms and cell death. Supported by SNF grant 310000–108223 and Eli Lilly Int. Found. grant to AE

Preferential 3-repeat tau staining of extracellular neurofibrillary tangles in Down syndrome with Alzheimer type changes

Preferential 3-repeat tau staining of extracellular neurofibrillary tangles in Down syndrome with Alzheimer type changes

Preferential 3‐repeat tau staining of extracellular neurofibrillary tangles in Down syndrome with Alzheimer type changes

Preferential 3‐repeat tau staining of extracellular neurofibrillary tangles in Down syndrome with Alzheimer type changes

Increased level of active GSK‐3β in Alzheimer’s disease and accumulation in argyrophilic grains and in neurones at different stages of neurofibrillary degeneration

The somatodendritic accumulation of hyperphosphorylated tau proteins is an early event preceding the appearance of neurofibrillary tangles (NFT) in Alzheimer’s disease (AD) and might be necessary for their formation. Glycogen synthase kinase‐3β (GSK‐3β) is a physiological kinase for tau that generates many tau phosphorylation sites identified in NFT and in other tau‐positive inclusions. We have studied the cellular distribution and the expression of the active form of GSK‐3β (GSK‐3 pTyr216) in AD patients, in argyrophilic grain disease and in diffuse Lewy body disease. By Western blotting analysis, a significant increase in the level of GSK‐3 (pTyr216) was observed in the frontal cortex of AD patients. A population of neurones showed a somatodendritic accumulation of GSK‐3 (pTyr216) but not of the inactive form of GSK‐3β (GSK‐3 pSer9). Most of these GSK‐3 (pTyr216)‐positive cells were positive for six different phosphotau epitopes known to be generated by GSK‐3β. By using a quadruple labelling method using GSK‐3 (pTyr216) and phosphotau immunolabelling combined with Gallyas and DAPI staining, we examined neurones containing a somatodendritic GSK‐3 (pTyr216) immunoreactivity at different stages of neurodegeneration. A majority of neurones at the pretangle stage without Gallyas‐positive inclusions were GSK‐3 (pTyr216) positive and this GSK‐3 (pTyr216) immunoreactivity remained in most cells containing Gallyas and phosphotau‐positive inclusions excepted in extracellular NFT. A GSK‐3 (pTyr216) immunoreactivity was present in argyrophilic grains but not in cortical Lewy bodies. These results directly suggest that the activity of GSK‐3β is increased in AD and that somatodendritic accumulation and activation of GSK‐3β is an early event preceding and accompanying the formation of NFT and of other tau‐positive inclusions.

Identification of AGE-modified proteins in SH-SY5Y and OLN-93 Cells

The formation of "Advanced Glycation End products" (AGEs) is an inevitable consequence of mammalian glucose metabolism. AGE-mediated protein-protein crosslinks lead to detergent-insoluble and protease-resistant protein aggregates, and in Alzheimer's disease (AD) extra cellular senile plaques (SPs) and intracellular neurofibrillary tangles (NFTs) have been shown to contain AGEs. However, to date little is known concerning the most prevalent protein-targets of AGE modification under normal, non-pathological conditions. Here, a combination of 2D-electrophoresis, Western blotting and mass spectrometry has been used to identify preferentially AGE-modified proteins in oligodendrocyte (OLN-93) and neuroblastoma cell lines (SH-SY5Y) in standard culture. Proteomics analysis identified a total of eight targets with structural, metabolic and regulatory function, three of which (beta-actin, beta-tubulin and eukaryotic Elongation Factor 1-alpha) were common to both cell lines. Based on results from prior studies, modification of these proteins may lead to a loss of function. Consequently, the identification of targets for these proteins is of particular interest for a better understanding of the consequences of AGE-modification in aging, neurodegenerative diseases and diabetes.

Aluminum in hippocampal neurons from humans with Alzheimer's disease

Using a staining technique developed in 2004, we examined hippocampal tissue from autopsy-confirmed cases of Alzheimer's disease (AD) and controls. The stain disclosed aluminum in cells and subcellular structure. All pyramidal neurons in these aged specimens appeared to exhibit at least some degree of aluminum staining. Many displayed visible aluminum only in their nucleolus. At the other extreme were neurons that stained for aluminum throughout their nucleus and cytoplasm. The remainder exhibited intermediate degrees of staining. On the basis of their aluminum staining patterns, all pyramidal neurons could be classified into stages that indicated two distinct neuropathological processes, either (1) progressive increase of nuclear aluminum (often accompanied by granulovacuolar degeneration with granules that stain for aluminum) or (2) formation of neurofibrillary tangles (NFTs) in regions of aluminum-rich cytoplasm, especially in AD brain tissue. In the latter process, intraneuronal NFTs appeared to displace nuclei and then enucleate the affected neurons during the course of their transformation into extracellular NFTs. Given that the NFTs we observed in human neurons always developed in conjunction with cytoplasmic aluminum, we hypothesize that aluminum plays an important role in their formation and should therefore be reconsidered as a causative factor for AD.

Relationship between microtubule-binding repeats and morphology of neurofibrillary tangle in Alzheimer's disease

The present study was performed to compare the distributions of three-repeat (3R) and four-repeat (4R) neurofibrillary tangles (NFT) with those of pretangles (p-NFT), intracellular NFT (i-NFT), and extracellular NFT (e-NFT) in the hippocampus of Alzheimer's disease brains. NFT labeling was performed using anti-tau antibodies: pSer262 for p-NFT, pSer422 for i-NFT, AT8 for e-NFT, RD3 for 3R, and RD4 for 4R tau, and Gallyas impregnation for the NFT population. RD4- and pSer422-positive NFT were detected predominantly in sectors from CA2 to CA4, while RD3- and pSer262-positive NFT were predominantly present in CA1, the entorhinal cortex, and the subiculum. The tau epitope recognized by pSer262 belongs to 4R tau but it showed a strong correlation with RD3- and AT8-positive NFT. Sectors CA2-CA4 showed predominantly 4R-NFT containing the pSer422 epitope. pSer262 may detect the process of transformation from p-NFT to i-NFT, and e-NFT consisted predominantly of 3R tau.

Similarities between CSF–brain extracellular transfer and neurofibrillary tangle invasion in Alzheimer's disease

Using the in vivo enzyme protection-enzyme inhibition method, we visualized the distribution of the intraventricularly and cisternally (cisterna magna) injected ambenonium chloride (Am) bound reversibly to the extracellular acetylcholinesterase enzyme (AChE) in the rabbit brain in order to describe the extracellular flow pathways from the cerebrospinal fluid (CSF). We found that the distribution of Am-protected AChE (indicating the Am itself) is similar to tracers having no intracerebral binding sites. The topographical distribution after both ways of application indicates a preferential penetration of Am into the limbic structures of the cerebral hemispheres in a predictable topographic sequence starting from the corticoid areas, allo- and periallo cortices followed by the mesocortical regions and then, in a limited extent, to the isocortex. The lentiform nuclei and the central part of diencephalic halves are inaccessible to Am. The hierarchic order in the sequence of diffusion from the CSF into the hemispheric subpial regions and the distribution pattern of Am resemble the stereotypic topographic expansion pattern and the predominantly limbic distribution of neurofibrillary tangles (NFTs) in Alzheimer's disease and related conditions.

Aberrant accentuation of neurofibrillary degeneration in the hippocampus of Alzheimer's disease with amyloid precursor protein 717 and presenilin-1 gene mutations

This study reports correlation of the hippocampal neurofibrillary tangles (NFT) density with beta-amyloid (Aβ) precursor protein (APP) 717 mutation, presenilin (PS)-1 mutation and apolipoprotein E (Apo-E) e4 alleles (E4), being graded as 3 forms (no-E4, one-E4 and two-E4) in autopsied brains from patients with familial and non-familial Alzheimer's disease (AD). We studied the density of NFT-free neurons, intracellular NFT (I-NFT), extracellular NFT (E-NFT) and total NFT (I-NFT plus E-NFT) in the six hippocampal subdivisions: cornu ammonis (CA) 1–CA4, subiculum and entorhinal cortex. The APP mutation cases showed significantly higher total NFT density in the CA1–CA2 region, and the PS-1 mutation cases also showed higher density of total NFT in the CA1–CA3 than non-familial cases. Moreover, high densities of the E-NFT contributed to these high total NFT densities. Non-familial AD cases showed a stereotypical NFT distribution with entorhinal accentuation in the hippocampus irrespective of E4 frequency. Thus, APP and PS-1 mutations predominantly affect the CA regions with profound neurodegeneration, which contributes early and severe clinical features of familial AD.

14-3-3 proteins and zeta isoform containing neurofibrillary tangles in patients with Alzheimer's disease.

Immunolocalization of 14-3-3 proteins in Alzheimer's disease (AD) brains was investigated using isoform-specific antibodies. Weak granular immunoreactivity of 14-3-3 proteins was found in neuronal cytoplasm in control subjects and AD brains. Both intracellular and extracellular neurofibrillary tangles (NFTs), as well as neuropil thread-like structures, were immunopositive for 14-3-3 proteins. This was corroborated by triple-fluorolabeling method visualizing paired helical filament (PHF) tau and 14-3-3 epitopes in relation to fibrillary state detected by thiazin red. Pretangle neurons (positive for PHF-tau without fibrillary structure detected by thiazin red) only contained fine granular immunoreactivity (IR) of 14-3-3, which was similarly found in unaffected neurons. Granular cytoplasmic IR of 14-3-3 proteins in pretangle neurons was not colocalized to granular tau-like IR, which suggests that participation of 14-3-3 proteins in NFT formation was restricted to its later stages. Its zeta isoform was most prominent in these NFTs, suggesting that this isoform is a major component involved in the formation of NFTs. In contrast, IR of epsilon isoform was found in the neuropil of the hippocampus and that of sigma isoform was localized to granule cells of the dentate gyrus in AD brains, as seen in the age-matched controls. Expression of 14-3-3 proteins were found to be highly variable and dependent on their isoforms, regions and cell types. Molecular, as well as topographical, dissection of 14-3-3 proteins will provide us with an improved understanding of this molecule in normal and pathological conditions.

Stereologic analysis of neurofibrillary tangle formation in prefrontal cortex area 9 in aging and Alzheimer’s disease

Alzheimer’s disease (AD) is characterized neuropathologically by several features including extensive neuronal death in the cerebral cortex. In fact, while neuropathological changes restricted to the hippocampal formation are a consistent reflection of age-related memory impairment, overt dementia is present only in cases with neocortical involvement. Several quantitative studies have reported a substantial loss of neurons from these regions and a parallel increase in the number of neurofibrillary tangles (NFT). However, accurate quantitative data on the dynamics of NFT formation are lacking. In the present study, we performed a stereologic analysis of the proportions of intracellular and extracellular (ghost) NFT, and unaffected neurons in the deep part of layer III (layer IIIc) and the superficial part of layer V (layer Va) of Brodmann’s prefrontal cortex area 9. Elderly cognitively unimpaired cases were compared with cases with different degrees of cognitive dysfunction. The data revealed differential rates of formation of intracellular and extracellular NFT between the two layers, and confirmed the presence of a severe disease-associated, but not age-related, neuronal loss. It was also shown that a susbtantial number of pyramidal cells may persist either unaffected or in a transitional stage of NFT formation in both neocortical layers. These results suggest that a considerable number of neurons containing an intracellular NFT exists in the neocortex until late in the course of AD. Whereas it is not possible to assess whether such transitional neurons are fully functional, these affected neurons might respond positively to therapeutic strategies aimed at protecting the cells that are prone to neurofibrillary degeneration in AD.

Stereologic evidence for persistence of viable neurons in layer II of the entorhinal cortex and the CA1 field in Alzheimer disease.

The entorhinal cortex and hippocampus are the first cortical regions to be affected by the degenerative cellular process that leads to Alzheimer disease (AD) and display a limited degree of neuronal alterations in normal aging. Several quantitative studies have reported a substantial loss of neurons in these regions and a parallel increase in the number of neurofibrillary tangles (NFTs). However, accurate quantitative data on the dynamics of NFT formation are lacking. Here, we performed a stereologic assessment of the proportions of intracellular and extracellular (ghost) NFTs (iNFTs and eNFTs, respectively) and unaffected neurons in layer II of the entorhinal cortex and in the pyramidal cell layer of the CA1 field of the hippocampus in elderly control cases compared to cases with varying degrees of cognitive dysfunction. The data revealed differential rates of formation of iNFTs and eNFTs between the 2 regions and confirmed the presence of a severe disease-associated, but not age-related, neuronal loss. They also revealed that large numbers of neurons may persist either unaffected or in a transitional stage of NFT formation until the late stages of AD progression. These neurons with viability potential constitute 73% of the total numbers of profiles in layer II of the entorhinal cortex and 77% in the CA1 field in cases with a Clinical Dementia Rating score of 3. Whereas it is not possible in the present study to assess how functional such neurons with altered physiology might be, it is nonetheless likely that these transitional neurons open new options for potential therapeutic interventions aimed at protecting neurons vulnerable to neurofibrillary degeneration.

Neuron loss from the hippocampus of Alzheimer's disease exceeds extracellular neurofibrillary tangle formation.

Neurofibrillary tangle (NFT) formation in the CA1 region of the hippocampus is one of the early events in the pathogenesis of Alzheimer's disease (AD). As the disease progresses more NFTs form and there is substantial neuron loss. In this study we investigated whether NFT formation accounts for all the CA1 pyramidal neuron loss seen in AD. Using unbiased stereological techniques, we estimated the total number of neurons and the number of intra- and extra-cellular NFTs in the hippocampus of 10 patients with AD and 10 age-matched controls. Marked neuronal loss (approximately 60%) was identified in AD, although NFTs accounted for only a small proportion of this loss (2.2-17.2%, mean 8.1%). Analysis of NFT accumulation with duration of dementia showed a linear relationship, supporting the belief that NFTs progressively accumulate with time.

Specific tau phosphorylation sites correlate with severity of neuronal cytopathology in Alzheimer's disease.

Microtubule associated protein tau is abnormally phosphorylated in Alzheimer's disease (AD) and aggregates as paired helical filaments (PHFs) in neurofibrillary tangles (NFTs). We show here that the pattern of tau phosphorylation correlates with the loss of neuronal integrity. Studies using 11 phosphorylation dependent tau antibodies and a panel of AD cases of varying severity were evaluated in terms of three stages of neurofibrillary tangle development: (1) pre-neurofibrillary tangle, (2) intra-, and (3) extra-neuronal neurofibrillary tangles. The pretangle state, in which neurons display nonfibrillar, punctate regions in the cytoplasm, sound dendrites, somas, and nuclei, was observed especially with phospho-tau antibodies TG3 (pT231), pS262, and pT153. Intraneuronal neurofibrillary tangles are homogenously stained with fibrillar tau structures, which were most prominently stained with pT175/181, 12E8 (pS262/pS356), pS422, pS46, pS214 antibodies. Extracellular NFTs, which contain substantial filamentous tau, are most prominently stained with AT8 (pS199/pS202/pT205), AT100 (pT212/pS214), and PHF-1 (pS396/pS404) antibodies, which also stain intracellular NFT. The sequence of early tau phosphorylation suggests that there are events prior to filament formation that are specific to particular phosphorylated tau epitopes, leading to conformational changes and cytopathological alterations.

Accumulation of C-terminally truncated tau protein associated with vulnerability of the perforant pathway in early stages of neurofibrillary pathology in Alzheimer's disease

Neurofibrillary pathology is a characteristic hallmark of Alzheimer's disease that is closely correlated with cognitive decline. We have analysed the density and distribution of neurofibrillary tangles (NFTs) that are immunoreactive with the monoclonal antibody (mAb) 423 in a prospectively analysed population of Alzheimer's disease (AD) cases and age-matched controls. NFTs were examined in allocortical and isocortical areas and correlated with Braak pathological stage and clinical severity of dementia. The mAb 423 was used as it recognises a C-terminally truncated tau fragment that is a major constituent of NFTs. Our results show that extracellular NFTs and, to a lesser extent, intracellular NFTs, correlated significantly with both Braak stages and the clinical index of severity. Furthermore, a differential distribution of the two types of tangles indicates that layer II of the entorhinal cortex and the transentorhinal area are particularly vulnerable to neurofibrillary degeneration. These areas serve as a point of connection between isocortex and hippocampus. Our findings, therefore, suggest that the perforant pathway may be substantially affected by the accumulation of truncated tau protein in AD and that this represents a neuropathological predictor for the clinical severity of dementia. When neurofibrillary pathology was examined by combined labelling with mAbs 423 and Alz-50 and the dye thiazin red, we were able to demonstrate various stages of tau aggregation. The different stages may represent a sequence of conformational changes that tau proteins undergo during tangle formation in the allocortex during the early development of dementia in AD.

The relationship between alphaB-crystallin and neurofibrillary tangles in Alzheimer's disease.

AlphaB-crystallin is known as a small heat shock protein with a cytoprotective function. This study was undertaken to investigate the relationship between alphaB-crystallin and changes seen in Alzheimer's disease. The distribution and immunohistochemical characteristics of alphaB-crystallin positive neurones in the cerebral cortices of 4 patients with Alzheimer's disease were examined. AlphaB-crystallin positive neurones were mainly distributed in the limbic and paralimbic regions, namely parahippocampal gyrus, fusiform gyrus, cingulate cortex, middle and superior frontal gyrus, and insular cortex, which corresponded to commonly affected regions in Alzheimer's disease. Moreover, such neurones were present predominantly in the III or V layer of the cerebral cortex. The number of alphaB-crystallin positive neurones increased in parallel with the neuronal loss. Logistic regression analysis revealed a significant relation between the density of alphaB-crystallin positive neurones and that of extracellular neurofibrillary tangles (NFTs), with a correlation coefficient (r) of 0.57 and P < 0.0001 in 14 regions of the cerebral cortex. In contrast, the relation was not statistically significant between the density of alphaB-crystallin positive neurones and that of classical senile plaques, diffuse plaques or intracellular NFTs. Modified Gallyas-Braak (GB) staining on alphaB-crystallin positive neurone demonstrated several patterns of the structures: faint GB positive structures in the swollen perikaryon with straight neurites, fine granules compressed and contorted into fuzzy bundles, intensely GB positive filamentous structures together with fine granules and very intensely GB positive ring-like NFTs in a swollen perikaryon with curved neurites. In positive neurones, the density of ring-like NFTs correlated with that of atrophic perikaryon, or bent neurites and a decrease in the immunoreactivity of alphaB-crystallin. These data suggest that a close relationship exists between the appearance of alphaB-crystallin in neurones, extracellular NFTs, and neurofibrillary formation in alphaB-crystallin positive neurones in Alzheimer brain.

Activation Mechanism of Brain Microglia in Patients With Diffuse Neurofibrillary Tangles With Calcification: A Comparison With Alzheimer Disease

Diffuse neurofibrillary tangles with calcification (DNTC) is an atypical dementia and is characterized pathologically by diffuse neurofibrillary tangles (NFTs) without senile plaques (SPs). In this study, we investigated the distribution of human leukocyte antigen (HLA)-DR-positive activated microglia in postmortem brain tissue of six patients with DNTC and six patients with Alzheimer disease (AD). HLA-DR-positive activated microglia were observed to associate with SPs in AD. In the DNTC brain, which lacks SPs, HLA-DR-positive microglia were mainly accumulated around weakly tau-positive NFTs, which were also positive for anti-amyloid-P and anti-C3d antibodies. The results of this study suggest that the complement pathway is also activated in the DNTC brain and that immune and inflammatory responses, including microglia activation, may occur around extracellular NFTs in DNTC patients.

A double-labeling immunohistochemical study of tau exon 10 in Alzheimer's disease, progressive supranuclear palsy and Pick's disease

Neurofibrillary tangles (NFT), one of the histopathological hallmarks of Alzheimer's disease (AD) and progressive supranuclear palsy (PSP), and Pick bodies in Pick's disease (PiD) are composed of microtubule-associated protein tau, which is the product of alternative splicing of a gene on chromosome 17. Alternative expression of exon 10 leads to formation of three- or four-repeat tau isoforms. To study the differential expression of exon 10, we performed double-labeling immunohistochemistry of the hippocampal formation in nine AD, four PSP and three PiD cases. Cryostat sections were processed with and without formic acid (FA) treatment, and double-stained with anti-tau (Alz-50 or PHF-1) or anti-amyloid P component antibodies and one of two specific anti-exon 10 antibodies (E-10). The effect of proteinase-K treatment was also evaluated. The results suggest the following. First, in AD, E-10 immunoreactivity is present in most intracellular NFT, but not in most dystrophic neurites and neuropil threads, suggesting differential expression of tau isoforms in specific cellular domains. Second, in AD, E-10 immunoreactivity is lost or blocked in most extracellular NFT, possibly due to proteolysis. Third, in PSP, E-10 immunoreactivity is hidden or blocked in NFT and tau-positive glial inclusions, but FA treatment exposes the epitope consistent with the hypothesis that PSP inclusions contain four-repeat tau. Fourth, E-10 immunoreactivity is present in dentate fascia NFT in AD and PSP, but not in Pick bodies in the dentate fascia or other areas. The results suggest that expression of exon 10 in tau is specific for cellular domains in a disease-specific manner.