Disease Senile Plaques Research Articles

Mechanisms of Protein Seeding in Neurodegenerative Diseases

Most age-associated neurodegenerative diseases involve the aggregation of specific proteins within the nervous system. In Alzheimer disease, the insidious pathogenic process begins many years before the symptoms emerge, and the lesions that characterize the disease—senile plaques and neurofibrillary tangles—ramify systematically through the brain. We review evidence that the -amyloid and tau proteins, which aggregate to form senile plaques and neurofibrillary tangles, respectively, are induced to misfold and self-assemble by a process of templated conformational change that amplifies a toxic species. Recent data also indicate that the spread of these lesions from one site to another is mediated by the cellular uptake, transport, and release of endogenous seeds formed by the cognate proteins. This simple pathogenic principle suggests that the formation, trafficking, and metabolism of pathogenic protein seeds are promising therapeutic targets for Alzheimer disease and other neurodegenerative disorders.

Small Amphipathic Molecules Modulate Secondary Structure and Amyloid Fibril-forming Kinetics of Alzheimer Disease Peptide Aβ1–42

Amyloid fibril formation is associated with a number of debilitating systemic and neurodegenerative diseases. One of the most prominent is Alzheimer disease in which aggregation and deposition of the Aβ peptide occur. Aβ is widely considered to mediate the extensive neuronal loss observed in this disease through the formation of soluble oligomeric species, with the final fibrillar end product of the aggregation process being relatively inert. Factors that influence the aggregation of these amyloid-forming proteins are therefore very important. We have screened a library of 96 amphipathic molecules for effects on Aβ(1-42) aggregation and self-association. We find, using thioflavin T fluorescence and electron microscopy assays, that 30 of the molecules inhibit the aggregation process, whereas 36 activate fibril formation. Several activators and inhibitors were subjected to further analysis using analytical ultracentrifugation and circular dichroism. Activators typically display a 1:10 peptide:detergent stoichiometry for maximal activation, whereas the inhibitors are effective at a 1:1 stoichiometry. Analytical ultracentrifugation and circular dichroism experiments show that activators promote a mixture of unfolded and β-sheet structures and rapidly form large aggregates, whereas inhibitors induce α-helical structures that form stable dimeric/trimeric oligomers. The results suggest that Aβ(1-42) contains at least one small molecule binding site, which modulates the secondary structure and aggregation processes. Further studies of the binding of these compounds to Aβ may provide insight for developing therapeutic strategies aimed at stabilizing Aβ in a favorable conformation.

Gas1 Interferes with AβPP Trafficking by Facilitating the Accumulation of Immature AβPP in Endoplasmic Reticulum-Associated Raft Subdomains

The amyloid-β protein precursor (AβPP) is a type I transmembrane protein that undergoes maturation during trafficking in the secretory pathway. Proper maturation and trafficking of AβPP are necessary prerequisites for AβPP processing to generate amyloid-β (Aβ), the core component of Alzheimer's disease senile plaques. Recently, we reported that the glycosylphosphatidylinositol (GPI)-anchored protein growth arrest-specific 1 (Gas1) binds to and interferes with the maturation and processing of AβPP. Gas1 expression led to a trafficking blockade of AβPP between the endoplasmic reticulum (ER) and the Golgi. GPI-anchored proteins can exit the ER by transiting through raft subdomains acting as specialized sorting platforms. Here, we show that Gas1 co-partitioned and formed a complex with AβPP in raft fractions, wherein Gas1 overexpression triggered immature AβPP accumulation. Pharmacological interference of ER to Golgi transport increased immature AβPP accumulation upon Gas1 expression in these raft fractions, which were found to be positive for the COPII protein complex component Sec31A, a specific marker for ER exit sites. Furthermore, a Gas1 mutant lacking the GPI anchor that could not transit through rafts was still able to form a complex with AβPP but did not lead to immature AβPP accumulation in rafts. Together these data show that Gas1 interfered with AβPP trafficking by interacting with AβPP to facilitate its translocation into specialized ER-associated rafts where immature AβPP accumulated.

Synthesis and Biological Evaluation of Analogues of a Novel Inhibitor of β-Amyloid Secretion

A drug library of 17,200 compounds was screened to select small molecules that inhibit the secretion of amyloid beta peptide (Abeta), the major component of Alzheimer disease senile plaques, from a human neuronal cell line. Twenty-nine hits were validated that decreased Abeta secretion by >40% at 10 microM, for a 0.17% hit rate. A lead hit was selected for further study based on its activity and low cytotoxicity, and it was found to inhibit Abeta secretion through activation of the alpha-secretase pathway. Twenty-four commercially available and 53 synthesized analogues were analyzed for activity. Selected analogues were evaluated for biological stability by incubation with hepatoma cells and for transcellular permeability using Caco-2 cell monolayers. The analogue with the best permeability was evaluated in 2-month old amyloid precursor protein transgenic mice and found to acutely reduce cerebral Abeta levels by 40% after a single iv administration.

Enrichment of cholesterol in microdissected Alzheimer’s disease senile plaques as assessed by mass spectrometry

Extensive knowledge of the protein components of the senile plaques, one of the hallmark lesions of Alzheimer's disease, has been acquired over the years, but their lipid composition remains poorly known. Evidence suggests that cholesterol contributes to the pathogenesis of Alzheimer's disease. However, its presence within senile plaques has never been ascertained with analytic methods. Senile plaques were microdissected from sections of the isocortex in three Braak VI Alzheimer's disease cases and compared with a similar number of samples from the adjoining neuropil, free of amyloid-beta peptide (A beta) deposit. Two cases were apo epsilon 4/apo epsilon 3, and one case was apo epsilon 3/apoepsilon3. A known quantity of (13)C-labeled cholesterol was added to the samples as a standard. After hexane extraction, cholesterol content was analyzed by liquid chromatography coupled with electrospray ionization mass spectrometry. The mean concentration of free cholesterol was 4.25 +/- 0.1 attomoles/microm(3) in the senile plaques and 2.2 +/- 0.49 attomoles/microm(3) in the neuropil (t = 4.41, P < 0.0009). The quantity of free cholesterol per senile plaque (67 +/- 16 femtomol) is similar to the published quantity of A beta peptide. The highly significant increase in the cholesterol concentration, associated with the increased risk of Alzheimer's disease linked to the apo epsilon 4 allele, suggests new pathogenetic mechanisms.

Enrichment of cholesterol in microdissected Alzheimer's disease senile plaques as assessed by mass spectrometry

Enrichment of cholesterol in microdissected Alzheimer's disease senile plaques as assessed by mass spectrometry

Zinc Dyshomeostasis, Ageing and Neurodegeneration: Implications of A2M and Inflammatory Gene Polymorphisms

Zinc maintains brain functions because involved in glutaminergic transmission, in antioxidant response and in conferring biological activity to brain enzymes and growth factors. Zinc turnover is mediated by Metallothioneins (MT) which regulate the intracellular free zinc ions [Zn](i). Alterations in zinc homeostasis are associated to various brain dysfunctions, including brain inflammatory status, but little is known about its implication in the aging brain and neurodegeneration. Literature data in experimental animals suggest that zinc dyshomeostasis may occur in aging associated to a decline in brain functions. One of the causes may be an altered homeostasis of MT and other zinc-binding proteins, such as alpha2 macroglobulin (A2M), which are of protection against stress and inflammation during young/adult age but turn into being harmful in aging. In fact, despite total brain zinc content is unchanged in the brain of aged animals, with respect to the young/adult, the activity of some zinc dependent enzymes is impaired and large amount of zinc has been found in the core of Alzheimer's disease senile plaques. The role played by MT and A2M is reported in ageing and Alzheimer's disease and on some polymorphisms of A2M and inflammatory genes (cytokines and their receptors) because some of them may be affected by zinc, via MT homeostasis.

A copper-binding site in the cytoplasmic domain of BACE1 identifies a possible link to metal homoeostasis and oxidative stress in Alzheimer's disease

The amyloidogenic processing pathway of the APP (amyloid precursor protein) generates Abeta (amyloid beta-peptide), the major constituent in Alzheimer's disease senile plaques. This processing is catalysed by two unusual membrane-localized aspartic proteinases, beta-secretase [BACE1 (beta-site APP-cleaving enzyme 1)] and the gamma-secretase complex. There is a clear link between APP processing and copper homoeostasis in the brain. APP binds copper and zinc in the extracellular domain and Abeta also binds copper, zinc and iron. We have found that a 24-residue peptide corresponding to the C-terminal domain of BACE1 binds a single copper(I) atom with high affinity through cysteine residues. We also observed that the cytoplasmic domain of BACE1 interacts with CCS, the dedicated copper chaperone for SOD1 (superoxide dismutase 1). Overproduction of BACE1 reduces SOD1 activity in cells. Consequently, SOD1 activity, cytosolic copper and ectodomain cleavage of APP are linked through BACE1.

Decay bounds in a model for aggregation of microglia: application to Alzheimer's disease senile plaques

We present a model for chemotaxis, as applied to the aggregation of microglia in Alzheimer's disease. Using biological parameters found in the literature, testable thresholds are derived such that amyloid plaques are predicted not to form if conditions fall below a threshold. The model we use was developed by Luca et al . (Luca et al . 2003 Bull. Math. Biol. 65 , 693–730) and incorporates terms for both attractant and repellent signals. Our analysis can be applied to both two and three-dimensional spatial domains with application to any cell system involving chemotaxis.

BACE1 Cytoplasmic Domain Interacts with the Copper Chaperone for Superoxide Dismutase-1 and Binds Copper

The amyloidogenic pathway leading to the production and deposition of Abeta peptides, major constituents of Alzheimer disease senile plaques, is linked to neuronal metal homeostasis. The amyloid precursor protein binds copper and zinc in its extracellular domain, and the Abeta peptides also bind copper, zinc, and iron. The first step in the generation of Abeta is cleavage of amyloid precursor protein by the aspartic protease BACE1. Here we show that BACE1 interacts with CCS (the copper chaperone for superoxide dismutase-1 (SOD1)) through domain I and the proteins co-immunoprecipitate from rat brain extracts. We have also been able to visualize the co-transport of membranous BACE1 and soluble CCS through axons. BACE1 expression reduces the activity of SOD1 in cells consistent with direct competition for available CCS as overexpression of CCS restores SOD1 activity. Finally, we demonstrate that the twenty-four residue C-terminal domain of BACE1 binds a single Cu(I) atom with high affinity through cysteine residues.

Chemotactic Signaling, Microglia, and Alzheimer’s Disease Senile Plaques: Is There a Connection?

Chemotactic cells known as microglia are involved in the inflammation associated with pathology in Alzheimer’s disease (AD). We investigate conditions that lead to aggregation of microglia and formation of local accumulations of chemicals observed in AD senile plaques. We develop a model for chemotaxis in response to a combination of chemoattractant and chemorepellent signaling chemicals. Linear stability analysis and numerical simulations of the model predict that periodic patterns in cell and chemical distributions can evolve under local attraction, long-ranged repulsion, and other constraints on concentrations and diffusion coefficients of the chemotactic signals. Using biological parameters from the literature, we compare and discuss the applicability of this model to actual processes in AD.

Exploring the Formation of Alzheimer's Disease Senile Plaques in Silico

An experimental simulation environment suitable for exploring the neuroinflammatory hypothesis of Alzheimer's disease (AD) has been developed. Using scientific literature, we have calculated parameters and rates and constructed an interactive model system. The simulation can be manipulated to explore competing hypotheses about AD pathology, i.e. can be used as an experimental “ in silico” system. In this paper, we outline the assumptions and aspects of the model, and illustrate qualitative and quantitative findings. The interactions of amyloid beta deposits, glial cell dynamics, inflammation and secreted cytokines, and the stress, recovery, and death of neuronal tissue are investigated. The model leads to qualitative insights about relative roles of the cells and chemicals in the disease pathology.

The disulphide bonds in the catalytic domain of BACE are critical but not essential for amyloid precursor protein processing activity.

beta-Site APP-cleaving enzyme (BACE) initiates the processing of the amyloid precursor protein (APP) leading to the generation of beta-amyloid, the main component of Alzheimer's disease senile plaques. BACE (Asp2, memapsin 2) is a type I transmembrane aspartic protease responsible for the beta-secretase cleavage of APP producing a soluble form of the ectodomain (sAPPbeta) and the membrane-bound, carboxy-terminal intermediates C99 and C89. BACE maturation involves cysteine bridge formation, N -glycosylation and propeptide removal. We investigated variants of BACE in which the disulphide bonds of the catalytic domain spanning between Cys216/Cys420, Cys278/Cys443 and Cys330/Cys380 were removed by mutagenesis. When transfected in cultured cells, these mutants showed impaired maturation. Nevertheless, a fraction of mutated protein retained both the competence to mature as well as the activity to process APP. For the generation of a functional enzyme the conserved Cys330/Cys380 bond was the most critical, whereas the two bonds between Cys216/Cys420 and Cys278/Cys443, which are typical for the membrane-bound BACE, appeared to be less important.

Expression of human β‐secretase in the mouse brain increases the steady‐state level of β‐amyloid

beta-Site APP-cleaving enzyme (BACE) initiates the processing of the amyloid precursor protein (APP) leading to the generation of beta-amyloid, the main component of Alzheimer's disease senile plaques. BACE (Asp2, memapsin 2) is a type I transmembrane aspartyl protease and is responsible for the beta-secretase cleavage of APP producing different endoproteolytic fragments referred to as the carboxy-terminal C99, C89 and the soluble ectodomain sAPPbeta. Here we describe two transgenic mouse lines expressing human BACE in the brain. Overexpression of BACE augments the amyloidogenic processing of APP as demonstrated by decreased levels of full-length APP and increased levels of C99 and C89 in vivo. In mice expressing huBACE in addition to human APP wild-type or carrying the Swedish mutation, the induction of APP processing characterized by elevated C99, C89 and sAPPbeta, results in increased brain levels of beta-amyloid peptides Abeta40 and Abeta42 at steady-state.

APP transgenic mice Tg2576 accumulate Abeta peptides that are distinct from the chemically modified and insoluble peptides deposited in Alzheimer's disease senile plaques.

The amyloid (Abeta) peptides generated in Hsiao's APP Tg2576 transgenic (Tg) mice are physically and chemically distinct from those characteristic of Alzheimer's disease (AD). Transgenic mouse Abeta peptides were purified using sequential size-exclusion and reverse-phase chromatographic systems and subjected to amino acid sequencing and mass spectrometry analyses. The mouse Abeta peptides lacked the extensive N-terminal degradations, posttranslational modifications, and cross-linkages abundant in the stable Abeta peptide deposits observed in AD. Truncated Abeta molecules appear to be generated in vivo by hydrolysis at multiple sites rather than by post-mortem C-terminal degradation. In contrast to AD amyloid cores, the Tg mice peptides were soluble in Tris-SDS-EDTA solutions, revealing both monomeric and SDS-stable oligomeric species of Abeta. In contrast to our report on Novartis Pharma APP23 Tg mice [Kuo et al. (2001) J. Biol. Chem. 276, 12991], which maintain high levels of soluble Abeta early on with later development of extensive vascular amyloid, Tg2576 mice exhibited an age-related elevation of soluble Abeta with relatively limited vascular amyloid deposition. The transgenic mouse levels of carboxy-terminal (CT) APP fragments were nearly 10-fold greater than those of human brains, and this condition may contribute to the unique pathology observed in these animals. Immunization of transgenic mice may act to prevent the pathological effects of betaAPP overproduction by binding CT molecules or halting their processing to toxic forms, in addition to having any effects on Abeta itself. Thus, differences in disease evolution and biochemistry must be considered when using transgenic animals to evaluate drugs or therapeutic interventions intended to reduce the Abeta burden in Alzheimer's disease.

A Splice Variant of β-Secretase Deficient in the Amyloidogenic Processing of the Amyloid Precursor Protein

beta-Secretase (BACE) initiates the amyloidogenic processing of the amyloid precursor protein leading to the generation of the beta-amyloid, the main component of Alzheimer's disease senile plaques. BACE is a type I transmembrane aspartyl protease of 501 amino acids. Here we describe a novel BACE mRNA lacking 132 base pairs that is expressed in the pancreas but not in the brain. Sequence alignment indicates that the deleted fragment matches the terminal two-thirds of exon 3. The new BACE variant is short of a 44-amino acid region located between the two catalytic aspartyl residues. Accordingly, a 50-kDa form of BACE (BACE457) is detected in the human pancreas. When expressed in cells, BACE457 colocalizes with the marker for the endoplasmic reticulum BiP. Moreover, BACE457 remains in a proenzymatic and endoglycosidase H-sensitive state, suggesting that its transport along the secretory pathway is blocked at the level of the endoplasmic reticulum. Notably, this novel form of BACE does not contribute to the processing of the amyloid precursor protein. Our findings suggest that tissue-specific splicing of the BACE mRNA may explain the observation that in the human pancreas robust transcription of the BACE gene does not translate into recovered enzymatic activity.

Predominance of neuronal mRNAs in individual Alzheimer's disease senile plaques

The sequestration of RNA in Alzheimer's disease (AD) senile plaques (SPs) and the production of intraneuronal amyloid-beta peptides (Abeta) prompted analysis of the mRNA profile in single immunocytochemically identified SPs in sections of AD hippocampus. By using amplified RNA expression profiling, polymerase chain reaction, and in situ hybridization, we assessed the presence and abundance of 51 mRNAs that encode proteins implicated in the pathogenesis of AD. The mRNAs in SPs were compared with those in individual CA1 neurons and the surrounding neuropil of control subjects. The remarkable demonstration here, that neuronal mRNAs predominate in SPs, implies that these mRNAs are nonproteinaceous components of SPs, and, moreover, that mRNAs may interact with Abeta protein and that SPs form at sites where neurons degenerate in the AD brain.

Predominance of neuronal mRNAs in individual Alzheimer's disease senile plaques

Predominance of neuronal mRNAs in individual Alzheimer's disease senile plaques

Copper, iron and zinc in Alzheimer's disease senile plaques.

Concentrations of copper (Cu), iron (Fe) and zinc (Zn) were measured in the rims and cores of senile plaques (SP) and in the neuropil of the amygdala of nine Alzheimer's disease (AD) patients and in the neuropil of the amygdala of five neurologically normal control subjects using micro particle-induced X-ray emission (micro-PIXE). Comparison of SP rim and core values revealed no significant differences between levels of Cu, Fe or Zn. Zinc and Fe in SP rims and cores were significantly elevated in AD compared with AD neuropil (P<0.05). Copper was significantly elevated (P<0.05) in the rim of SP compared with AD neuropil. Comparison of AD and control neuropil revealed a significant (P<0.05) elevation of Zn in AD subjects. The elevation of these elements in SP in AD is of interest in light of the observation that Cu, Fe and particularly Zn, can accelerate aggregation of amyloid beta peptide.

Human prion diseases: Possible new directions in prophylaxis and therapy

The human prion diseases, kuru, Creutzfeldt-Jakob disease, Gerstmann--Str~iussler-Scheinker syndrome (1), fatal familial insomnia (2), and atypical dementia with octapeptide inserts (3), are slow, degenerative, transmissible dementing, fatal amyloidoses of the central nervous system (CNS). The worldwide incidence is generally thought to be approximately one per million people per year. The normal human prion protein, designated by PrP c, is encoded by a gene on chromosome 20. PrP C appears necessary for normal CNS synaptic function and is also expressed peripherally, although its function in the periphery is unknown. It is thought, however, to be involved in cell signalling and adhesion (4). PrP c has a very low beta sheet content, but is convertible by a conformational change to a protease resistant form denoted by PrP sC. This has a high beta sheet content, prpsC can aggregate to form brain amyloid deposits resistant to degradation (1). Current hypotheses suggest that the spreading pathology of prion diseases is due to recruitment of prpSC from the PrP c of host brain cells. But exactly how does this spreading pathology proceed? Some clues might be gained from the example of Alzheimer disease. The amyloid of Alzheimer disease starts with the extracellular deposition of ~-amyloid protein (BAP), thought to be derived from amyloid precursor protein produced in neurons. BAP can activate complement (5), and the activated fragments richly decorate senile plaques (6). Activated complement components can cause autodestruction of host tissue by generation of the membrane attack complex (6) and by stimulating microglial cells to generate toxic oxygen free radicals (7). Activated microglial cells are embedded in Alzheimer disease senile plaques and are well positioned to carry out such an attack (6). Degeneration of neuronal processes by these mechanisms can generate more extracellular BAP, resulting in an everexpanding autodestructive cycle. Anti-inflammatory agents appear capable of arresting this cycle. Multiple epidemiological studies now indicate that patients taking anti-inflammatory drugs, or suffering from diseases such as arthritis, in which such drugs are widely used, are relatively spared from Alzheimer disease (8-11). In a pilot clinical trial, indomethacin caused an apparent arrest of disease progression (12). The amyloid deposits of kuru, Creutzfeldt-Jakob disease and the Gerstmann-Striiussler-Scheinker syndrome have been reported to be associated with microglial cells (13-15). The presence of complement proteins in Creutzfeldt-Jakob amyloid deposits has also been reported (16). Taken together, these data suggest that a somewhat analogous autodestructive process may be occurring in these prion-associated disorders. Activation of complement would result in generation of the membrane attack complex as well as toxic oxygen species. The point of attack on neurons could be different from that in Alzheimer disease, result-