Amyloid Fibrils In Senile Plaques Research Articles

Bioinformatic analysis of the SPs and NFTs proteomes unravel putative biomarker candidates for Alzheimer's disease.

Aging is the main risk factor for the appearance of age-related neurodegenerative diseases, including Alzheimer's disease (AD). AD is the most common form of dementia, characterized by the presence of senile plaques (SPs) and neurofibrillary tangles (NFTs), the main histopathological hallmarks in AD brains. The core of these deposits are predominantly amyloid fibrils in SPs and hyperphosphorylated Tau protein in NFTs, but other molecular components can be found associated with these pathological lesions. Herein, an extensive literature review was carried out to obtain the SPs and NFTs proteomes, followed by a bioinformatic analysis and further putative biomarker validation. For SPs, 857 proteins were recovered, and, for NFTs, 627 proteins of which 375 occur in both groups and represent the common proteome. Gene Ontology (GO) enrichment analysis permitted the identification of biological processes and the molecular functions most associated with these lesions. Analysis of the SPs and NFTs common proteins unraveled pathways and molecular targets linking both histopathological events. Further, validation of a putative phosphotarget arising from the in silico analysis was performed in serum-derived extracellular vesicles from AD patients. This bioinformatic approach contributed to the identification of putative molecular targets, valuable for AD diagnostic or therapeutic intervention.

Mechanism of Cellular Formation and In Vivo Seeding Effects of Hexameric \u03b2-Amyloid Assemblies

The β-amyloid peptide (Aβ) is found as amyloid fibrils in senile plaques, a typical hallmark of Alzheimer’s disease (AD). However, intermediate soluble oligomers of Aβ are now recognized as initiators of the pathogenic cascade leading to AD. Studies using recombinant Aβ have shown that hexameric Aβ in particular acts as a critical nucleus for Aβ self-assembly. We recently isolated hexameric Aβ assemblies from a cellular model, and demonstrated their ability to enhance Aβ aggregation in vitro. Here, we report the presence of similar hexameric-like Aβ assemblies across several cellular models, including neuronal-like cell lines. In order to better understand how they are produced in a cellular context, we investigated the role of presenilin-1 (PS1) and presenilin-2 (PS2) in their formation. PS1 and PS2 are the catalytic subunits of the γ-secretase complex that generates Aβ. Using CRISPR-Cas9 to knockdown each of the two presenilins in neuronal-like cell lines, we observed a direct link between the PS2-dependent processing pathway and the release of hexameric-like Aβ assemblies in extracellular vesicles. Further, we assessed the contribution of hexameric Aβ to the development of amyloid pathology. We report the early presence of hexameric-like Aβ assemblies in both transgenic mice brains exhibiting human Aβ pathology and in the cerebrospinal fluid of AD patients, suggesting hexameric Aβ as a potential early AD biomarker. Finally, cell-derived hexameric Aβ was found to seed other human Aβ forms, resulting in the aggravation of amyloid deposition in vivo and neuronal toxicity in vitro.

Characterization of the unique In Vitro effects of unsaturated fatty acids on the formation of amyloid β fibrils.

Accumulation of amyloid ß (Aß) peptides, the major component of amyloid fibrils in senile plaques, is one of the main causes of Alzheimer’s disease. Docosahexaenoic acid (DHA) is a fatty acid abundant in the brain, and is reported to have protective effects against Alzheimer’s disease, although the mechanistic effects of DHA against Alzheimer’s pathophysiology remain unclear. Because dietary supplementation of DHA in Aß precursor protein transgenic mice ameliorates Aß pathology and behavioral deficits, we hypothesize that DHA may affect the fibrillization and deposition of Aß. Here we studied the effect of different types of fatty acids on Aß fibril formation by in vitro Aß fibrillization assay. Formation of amyloid fibrils consists of two steps, i.e., the initial nucleation phase and the following elongation phase. We found that unsaturated fatty acids, especially DHA, accelerated the formation of Aß fibrils with a unique short and curved morphology in its nucleation phase, which did not elongate further into the long and straight, mature Aß fibrils. Addition of DHA afterwards did not modify the morphology of the mature Aß(1–40) fibrils. The short and curved Aß fibrils formed in the presence of DHA did not facilitate the elongation phase of Aß fibril formation, suggesting that DHA promotes the formation of “off-pathway” conformers of Aß. Our study unravels a possible mechanism of how DHA acts protectively against the pathophysiology of Alzheimer’s disease.

Single Molecule Studies of Early Stages of Amyloid Aggregation an Effect of pH

The deposition of amyloid fibrils in senile plaques of the brain is the hallmark for many neurodegenerative diseases including Alzheimer's disease (AD). The normal pH in the neuron is neutral, which may decrease due to the mental depression or other environmental factors that may affect amyloid aggregation inside the brain. Moreover, liposomes in the brain are acidic. In vitro studies indicate that acidic pH accelerates amyloid aggregation but the molecular mechanism underlying this effect is unknown. In this work, we probed a Aβ(14-23) trimer and tetramer using our previously developed flexible nano array (FNA) technique to characterize the assembly of this peptide in trimers and tetramers depending on pH. Using atomic force microscopy-single molecule force spectroscopy, and fluorescence based tethered approach for probing intermolecular interactions (TAPIN) approach, we found that pH affects the pathways of oligomers assembly. If at neural pH there is primarily one assembly pathway, at acidic pH three different pathways with different stabilities were identified. Models for the assembly of Aβ(14-23) trimers and tetramers are proposed. We posit that the highly stable oligomers found at acidic pH lead to acceleration of aggregation process suggesting that acidic pH may be a risk factor in development of AD.Acknowledgements. The work was supported by NIH grants R01 GM096039 and R01GM118006 to YLL.

Bcl-2 and Bax protein expression in Alzheimer's disease.

Beta-amyloid deposition and neurofibrillary degeneration are important pathological findings in the brains of patients with Alzheimer's disease (AD). In the present study, we have examined Bcl-2 and Bax immunoreactivity in the hippocampus of AD cases, with special attention to the possible relationship between Bcl-2 and Bax immunoreactivity, and neurofibrillary degeneration and senile plaques. Different antibodies were used, including Bcl-2 (N-19), Bcl-2 (BioGenex), Bax (P-19) and Bax (N-20), and their specificity was tested on Western blots of brain homogenates. No differences between Bcl-2 and Bax immunoreactivity in tangle-bearing and non-tangle-bearing neurons were observed, thus suggesting that Bcl-2 and Bax do not participate in tangle formation. Overexpression of Bcl-2 protein in reactive glial cells surrounding senile plaques suggests that Bcl-2 may play a role in the survival of reactive glia. On the other hand, overexpression of Bax immunoreactivity in dystrophic neurites of senile plaques suggests that Bax is associated with neurite degeneration in senile plaques. Finally, Bax (P-19), but not Bax (N-20), immunoreactivity was localized in amyloid fibrils of senile plaques. Since Western blots to Bax (P-19) recognize multiple bands in addition to the expected band of about 21 kDa, it is suggested that Bax (P-19) immunoreactivity of amyloid fibrils is not specific.

Aluminum modifies the properties of Alzheimer's disease PHF tau proteins in vivo and in vitro

Hyperphosphorylated adult human CNS tau (PHF tau or A68) forms paired helical filaments (PHFs) in neurofibrillary tangles (NFTs), neuropil threads, and dystrophic neurites associated with senile plaques (SPs) during the progression of Alzheimer's disease (AD). While amyloid fibrils in SPs are composed of beta-amyloid (A beta), NFTs and SPs contain similar associated components such as ubiquitin, alpha 1-antichymotrypsin (ACT), apolipoprotein E (ApoE), heparan sulfate proteoglycans (HSPGs), and aluminum salts. Thus, SPs and NFTs may result from specific interactions between PHF tau, A beta, and these other components. In fact, intracerebral injections of PHF tau induce co-deposits of A beta, ACT, and ubiquitin (Shin et al., 1993). To examine this issue further, we probed interactions between PHF tau, aluminum salts, and other plaque and tangle components. We investigated in vivo interactions of PHF tau and aluminum chloride (AlCl3) with other plaque and tangle components by injecting PHF tau with and without AlCl3 into the rodent brain. PHF tau co-injected with AlCl3 formed aggregates that persisted much longer in the rat brain, and induced longer-lived co-deposits of A beta, ubiquitin, ACT, and ApoE than PHF tau alone. Injections of PHF tau with AlCl3 also induced neurons near the injection site to acquire PHF tau-like properties as monitored with antibodies (AT8, T3P, PHF1) that recognize defined PHF tau epitopes containing phosphoserine residues (Ser202, Ser396, Ser404). Injections of AlCl3 alone as well as injections of normal adult and fetal CNS tau, several different synthetic peptides, neurofilament proteins, ACT, HSPGs, or ApoE with and without AlCl3 failed to induce co-deposits of A beta or alter the immunoreactivity of tau in rodent neurons. To determine if aluminum salts interact directly and specifically with PHF tau in situ, we pretreated sections of AD hippocampus with 10 mM AlCl3 and then probed these sections by immunohistochemistry with antibodies to PHF tau as well as to a number of other plaque and tangle components. Preincubation of these sections with AlCl3 diminished PHF tau immunoreactivity in NFTs and SPs using the PHF tau-specific antibodies AT8, T3P, and PHF1, while the immunoreactivity of other plaque and tangle proteins (A beta, ubiquitin, ACT, HSPGs, ApoE) was not abolished. We also examined the effects of AlCl3 on PHF tau and normal adult human CNS tau in vitro. AlCl3 had no effect on normal adult human CNS tau, while increasing concentrations of AlCl3 (from 0.1 to 1.0 mM) induced PHF tau to aggregate at the top of the stacking gel, and at high concentrations (0.3 and 1.0 mM) of AlCl3, PHF tau completely failed to enter the gel. These studies suggest that aluminum binds to PHF tau, induces these proteins to aggregate, and retards their proteolysis. Further, since intracerebral injections of PHF tau with and without AlCl3 in rats appear uniquely capable of inducing co-deposits of a number of proteins found in authentic AD SPs and NFTs (including A beta, ubiquitin, ACT, and ApoE), we speculate that the contributions of PHF tau to plaque and tangle formation in AD may be modulated by aluminum.

A correlative study of the aluminum content and aging changes of the brain in non-demented elderly subjects

The interrelation of the aluminum content and aging change of the nerve cells was studied with special reference to the neurofibrillary tangle (NFT) and occurrence of senile plaque (SP), in 15 autopsied brains (cerebrum) of non-demented elderly subjects varying in age from 75 to 101 years old and 10 cases of non-demented non-elderly adult subjects. 1) In the brain of non-demented non-elderly adult subjects, the aluminum content of the hippocampus was 14.4 +/- 1.39 ppb and that of the cortex of the frontal lobe was 20.4 +/- 2.54 ppb. In the brain of the non-demented elderly subjects, the aluminum content of the hippocampus was 401.7 +/- 60.05 ppb and that of the cortex of the frontal lobe was 373.29 +/- 72.35 ppb. Comparison of the two groups showed obviously higher levels in the latter group, being 28 times in the hippocampus and 19 times in the cortex of the frontal lobe. 2) The histochemical demonstration of the aluminum localization by the Morin method disclosed the positive fluorescence reaction along the part of the disintegrating neurofibrillary tangle and amyloid core in the senile plaques of non-demented elderly subjects. In the normal brain tissue, positive fluorescence reaction was observed in the wall of the capillary vessels of the blood brain barrier, perivascular glial supporting tissues, the nuclei of the astrocytes and nuclei, nucleoli of the nerve cells partially. No fluorescence reaction was observed in the non-demented adult subjects. 3) Morphologically, the appearance of the neurofibrillary tangle was noted in some cases of non-demented elderly group with higher aluminum levels. Classical and diffuse type senile plaques were dispersed in the 1st to 4th zone of the cortex of the frontal lobe. However, diffuse type senile plaques in the hippocampus appeared to a lesser extent. Neurofibrillary tangles were slightly noted in the cortex of the frontal lobe, however marked increase was confirmed in the CA1 to CA3 zones of the hippocampus. Ultrastructurally, paired helical filaments (PHF) were noted in the neurofibrillary tangle constituent and the increase of amyloid fibrils in senile plaques was confirmed. In this study, high levels of the aluminum content of the brain were confirmed in the non-demented elderly subjects. Moreover, the histochemical demonstration of aluminum localization by the Morin method disclosed a positive fluorescence reaction along the sites of neurofibrillary tangles and amyloid core in the senile plaques of non-demented elderly subjects.(ABSTRACT TRUNCATED AT 400 WORDS)

Amyloid-P-component-like immunoreactivity in beta/A4-immunoreactive deposits in Alzheimer-type dementia brains.

An immunohistochemical study using the mirror-image technique was performed in order to establish whether amyloid P component is involved in the mechanism of deposition of amyloid fibrils in senile plaques (SPs) in Alzheimer-type dementia (ATD). Ninety percent of beta/A4 protein-immunoreactive SPs were also stained by the anti-amyloid P component immunohistochemistry, and this applied to all of the diffuse, primitive and classical types of beta/A4 deposits. These findings may suggest an involvement of amyloid P component in the formation of amyloid fibrils in senile plaques in ATD brains.

Amyloid protein in Alzheimer's disease.

Progressive deposition of amyloid fibrils in senile plaques and in blood vessels is a pathological hallmark of Alzheimer's disease. The AD amyloid protein, called beta amyloid protein, or A4 protein, is derived from a much larger precursor protein, the gene for which has now been cloned, sequenced, and localized to chromosome 21. This chromosomal location is of great interest because it has long been known that all Down's patients over the age of 40 develop the classical neuropathological lesions of AD. An anonymous DNA marker, which segregates with cases of dominantly inherited AD, has also been found to be located on chromosome 21. It is now known, however, that this marker and the gene encoding the beta amyloid precursor protein are not tightly linked. The beta amyloid precursor protein gene appears to code for a normal cellular product whose function is not yet known. The gene is expressed not only in brain but also in many non-neural tissues. It is highly conserved in evolution. Two closely related alternative transcripts have recently been identified; these contain an insert showing homology to certain members of the Kunitz family of proteinase inhibitors. All evidence accumulated thus far suggests that the beta amyloid precursor protein gene is not abnormal in AD; therefore, recent research has focused on transcriptional, translational, or posttranslational events that may be implicated in the progressive deposition of beta amyloid protein in AD.

An immunohistochemical study of ?-protein in Alzheimer-type dementia brains

An immunohistochemical study with a polyclonal anti-beta-protein antiserum was performed in order to understand the mechanism of deposition of amyloid fibrils in senile plaques in Alzheimer-type dementia (ATD). Serial cortical sections cut from ATD brains were necessary to investigate the structural correlation between senile plaques and blood vessels. The senile plaques were stained well and a blood vessel or capillary-like structure was found in most of beta-protein-immunoreactive deposits. These findings may suggest an involvement of blood vessels in the formation of at least some of the amyloid deposits in ATD brains.

Immuno-electron-microscopic localization of complements in amyloid fibrils of senile plaques.

The presence of components of immunoglobulins (Ishii et al. 1975) and complements (Eikelenboom and Stam 1982) in senile plaques suggests that the immunologic mechanisms are involved in the causation of pathologic processes in the brain of patients with Alzheimer's disease. Senile plaques consists of amyloid degenerated neuritis and glia, and exact localization of complements among these tissue elements will provide an important clue to the pathogenesis of the Alzheimer brain. This report deals with light- and electron-microscopic localization of complements in amyloid fibrils of senile plaques by immunoperoxidase histochemistry. The presence of C1q, C4, and C3 is confirmed light-microscopically. At the ultrastructural level, anti-complement C1q, C4, and C3 peroxidase reaction products are exclusively localized on the amyloid fibrils, but no other tissue elements, such as normal or degenerated neurites, neurofibrillary tangles, or glia. The results indicate the presence of immune complex in amyloid fibrils of senile plaques, and little association of complements in senile plaques with neurofilament protein.

Immuno-electron microscopic localization of immunoglobulins in amyloid fibrils of senile plaques.

Amyloid fibrils in senile plaques of the brain of patients with senile dementia or Alzheimer's disease combined specifically with horseradish peroxidase (HRPO)-labeled rabbit anti-human IgG. Light and electronmicroscopic immunoperoxidase technique was used to prove this. The fact may mean that immunological factors were involved in the pathogenesis of amyloid fibrils in the senile plaques, and probably also in that of senile dementia or Alzheimer's disease.