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Inflammation is believed to be integral to the pathogenesis of Alzheimer's disease (AD). Arachidonic acid (AA) is the most important omega-6 fatty acid and a mediator of inflammatory pathways. High-sensitivity enzyme linked immunosorbent assay shows that AA and its various metabolites; prostaglandins, thromboxanes, and leukotriene B4 resulted in significantly higher secretion of both Abeta40 and 42 peptides. A combination of identical number of alternate cis and trans double bonds either at positions Δ5 or 7Z,13 or 15E (such as PGE(2), PGF(2α), THXB2 and PGF(2α)EA) or at positions Δ6Z,8E,10E,14Z (such as LB4) built in the 3-dimensional structure of 20-carbon fatty acyl chains believed to be responsible for their detrimental action. CP 24,879 and sesamin, 2 inhibitors of the AA pathway suppressed the production of amyloid-beta (Aβ) peptides. Immunoblotting experiments and use of SP-C99 transfected COS-7 cells suggested that AA and its metabolites-driven altered production of Aβ is mediated through gamma-secretase cleavage of amyloid precursor protein (APP). An early-onset AD transgenic mouse model expressing the double-mutant form of human amyloid precursor protein, Swedish (K670N/M671L) and Indiana (V717F), corroborated our in vitro findings by showing higher levels of Abeta and amyloid plaques in the brains, when they were fed chow supplemented with 2% AA. Our work not only supports that AA and its metabolites are involved in the production of Aβ and in the pathogenesis of AD but also contributes to clarify aspects of structure-activity relationship helpful for future nonsteroidal anti-inflammatory drugs (NSAIDs) research., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Alzheimer's disease (AD) is the most common form of dementia. Amyloid-beta (A beta) peptide, generated by proteolytic cleavage of the amyloid precursor protein, is central to AD pathogenesis. Most pharmaceutical activity in AD research has focused on A beta, its generation and clearance from the brain. In particular, there is much interest in immunotherapy approaches with a number of anti-A beta antibodies in clinical trials. We have developed a monoclonal antibody, called WO2, which recognises the A beta peptide. To this end, we have determined the three-dimensional structure, to near atomic resolution, of both the antibody and the complex with its antigen, the A beta peptide. The structures reveal the molecular basis for WO2 recognition and binding of A beta. The A beta peptide adopts an extended, coil-like conformation across its major immunodominant B-cell epitope between residues 2 and 8. We have also studied the antibody-bound A beta peptide in the presence of metals known to affect its aggregation state and show that WO2 inhibits these interactions. Thus, antibodies that target the N-terminal region of A beta, such as WO2, hold promise for therapeutic development.

Cholesterol is implicated to play a role in Alzheimer disease pathology. Therefore, the concentrations of cholesterol, its precursors, and its degradation products in brain homogenates of aging wild-type and beta-amyloid precursor protein transgenic mice carrying the Swedish mutation (APP23) were analyzed. Among the sterols measured, lanosterol is the first common intermediate of two different pathways, which use either desmosterol or lathosterol as the predominant precursors for de novo synthesis of brain cholesterol. In young mice, cholesterol is mainly synthesized via the desmosterol pathway, while in aged mice, lathosterol is the major precursor. 24S-hydroxycholesterol (cerebrosterol), which plays a key role in the removal of cholesterol from the brain, modestly increased during aging. No differences in the levels of cholesterol, its precursors, or its metabolites were found between wild-type and APP23 transgenic mice. Moreover, the levels of the exogenous plant sterols campesterol and sitosterol were significantly elevated in the brains of APP23 animals at age 12 and 18 months. This time point coincides with abundant plaque formation.

Glucoconjugates of (+/-)-ibuprofen, (+/-)-alpha-tocopherol (vitamin E), gentisic acid, gallic acid, 2,6-bis(tert-butyl)-4-thiophenol, and N-acetyl-L-cysteine were prepared with the objective of increasing the bioavailability of such antioxidant and anti-inflammatory drugs. The O-glucosides were synthesized using benzylated alpha-D-glucopyranosyl trichloracetimidate as glycosyl donor. For the synthesis of the S-glucosides, the glycosyl donor 1,2,3,4,6-penta-O-acetyl-beta-D-glucopyranose provided higher yields than the corresponding O-acetylated imidate.

The prion protein (PrP) has a central role in the pathogenesis of transmissible spongiform encephalopathies (TSE). Accumulating evidence suggests that normal cellular PrP (PrP(c)) may be involved in copper homeostasis and modulation of copper/zinc superoxide dismutase (Cu/ZnSOD) activity in neurons. Hydrogen peroxide (H(2)O(2)) is a toxic reactive oxygen species generated through normal cellular respiration, and neurons contain two important peroxide detoxifying systems (glutathione pathway and catalase). To determine whether PrP expression affects neuronal resistance to H(2)O(2), we exposed primary cerebellar granule neuron cultures derived from PrP knockout (PrP(-/-)) and wild-type (WT) mice to H(2)O(2) for 3, 6, and 24 hours. The PrP(-/-) neurons were significantly more susceptible to H(2)O(2) toxicity than WT neurons after 6 and 24 hours' exposure. The increased H(2)O(2) toxicity may be related to a significant decrease in glutathione reductase activity measured in PrP(-/-) neurons both in vitro and in vivo. This was supported by the finding that inhibition of GR activity with 1,3-bis(2-chloroethyl)-1-nitrosurea (BCNU) increased H(2)O(2) toxicity in WT neurons over the same exposure period. The PrP toxic peptide PrP106-126 significantly reduced neuronal glutathione reductase activity and increased susceptibility to H(2)O(2) toxicity in neuronal cultures suggesting that PrP toxicity in vivo may involve altered glutathione reductase activity. Our results suggest the pathophysiology of prion diseases may involve perturbed PrP(c) function with increased vulnerability to peroxidative stress.

alpha-Synuclein (alphaSN), also termed the precursor of the non-Abeta component of Alzheimer's disease (AD) amyloid (NACP), is a major component of Lewy bodies and Lewy neurites pathognomonic of Parkinson's disease (PD) and dementia with Lewy bodies (DLB). A fragment of alphaSN termed the non-Abeta component of AD amyloid (NAC) had previously been identified as a constituent of AD amyloid plaques. To clarify the relationship of NAC and alphaSN with Abeta plaques, antibodies were raised to three domains of alphaSN. All antibodies produced punctate labeling of human cortex and strong labeling of Lewy bodies. Using antibodies to alphaSN(75-91) to label cortical and hippocampal sections of pathologically proven AD cases, we found no evidence for NAC in Abeta amyloid plaques. Double labeling of tissue sections in mixed DLB/AD cases revealed alphaSN in dystrophic neuritic processes, some of which were in close association with Abeta plaques restricted to the CA1 hippocampal region. In brain homogenates alphaSN was predominantly recovered in the cytosolic fraction as a 16-kd protein on Western analysis; however, significant amounts of aggregated and alphaSN fragments were also found in urea extracts of SDS-insoluble material from DLB and PD cases. NAC antibodies identified an endogenous fragment of 6 kd in the cytosolic and urea-soluble brain fractions. This fragment may be produced as a consequence of alphaSN aggregation or alternatively may accelerate aggregation of the full-length alphaSN.

In order to develop a system which allows infection by an epitope-specific phage-antibody via an F-pilus expressing that epitope, a study on the expression of foreign sequences on F-pilin was undertaken. Initially, a plasmid library was constructed with random sequences encoding one to five amino acid residues fused to the C terminus of F-pilin (traA) which was used to complement an F-plasmid with an amber mutation in traA. Functional F-pilin fusions were detected using the filamentous phage, fUSE2, which transduces tetracycline resistance, as well as immunoblots using a monoclonal antiserum specific for the acetylated N terminus of pilin. All the clones selected expressed the pilin-fusions and displayed full sensitivity towards fUSE2 infection, which was indistinguishable from the wild-type F-pilin. The sequences of fUSE2-sensitive clones when compared to randomly selected clones which were not fUSE2-sensitive, revealed no obvious pattern in the amino acid residues fused to the C terminus, except for a preference for a hydrophilic amino acid at position +1. Mutating the C-terminal Leu in wt (wild-type) pilin to Ser blocked pilus assembly and fUSE2 infection; the pilin was correctly processed but the level of acetylation at the N terminus appeared to decrease. Fusing a known epitope (myc) directly to the C terminus blocked processing of F-pilin leading to loss of F-pilus assembly and function. The introduction of random sequences between traA and this epitope yielded fully recombinant, functional F-pili but this appeared to be due to processing of the extension by an unidentified protease leading to loss of the epitope. Surface expression of another epitope (G2-10) was clearly demonstrated by immuno-electron microscopy of pili with a G2-10 monoclonal antibody. A different five amino acid residue spacer between the F-pilin C terminus and the G2-10 epitope produced a system that was transfer-proficient and fUSE2-sensitive, but the pili were barely detectable by immunoblots or by electron microscopy. While the underlying rules that govern successful epitope expression at the C terminus of F-pilin remain elusive, many types of foreign sequences can be displayed with varying degrees of success. Our results also suggest that pilin sequence determines a number of steps in the complex pathway for pilus assembly.

The consensus recommendations for the post mortem diagnosis Alzheimer's disease (AD) highlight the difficulties in establishing a pathological diagnosis in brains from clinically demented individuals with both certainty and uniformity. There is, however, a need for diagnostic guidelines that are relatively simple, inexpensive, and adaptable to general pathologists and different laboratories. The current Consortium to Establish a Registry for Alzheimer's disease (CERAD) criteria and the recommendations in the consensus document giving three probabilistic categories for diagnosis go a long way towards establishing a uniform approach for the diagnosis of AD. However, more uniformity could be adopted in the topography of sectioning to enhance diagnostic and future research comparisons. We also recommend that immunohistochemistry for beta A4 (A beta) amyloid and tau-reactive neurofibrillary changes, in addition to hematoxylin and eosin stains, should become the basis for histological diagnosis. We agree with the guidelines concerning documentation of all AD changes. Until a clearer understanding of the early changes of AD is established, strict observation and recording are the pathologists' best diagnostic skills. The ill-defined diagnostic areas of AD continue to prompt the need for a new method of detection of the underlying pathologic process.

The amyloid protein precursor (APP) of Alzheimer's disease (AD) is abundantly expressed in platelets, where its primary function remains undetermined. As an integral transmembrane protein, the release of APP from the membrane may be a critical event in AD. We examined the association of APP with human platelet membranes using a combination of alkali treatment and immunoprecipitation of the carboxyl-terminus of APP. Most of the platelet membrane-associated APP (APPMem) with molecular mass of 100 to 130 kD is removed with alkali treatment and is also truncated at the carboxyl-terminus. APPMem is present at least in part on the surface of the platelet. The full-length transmembrane form of APP, as a 140- to 150-kD minor species, is alkali resistant and is also present on the plasma membrane. In contrast, most of the APPMem from brain is full-length (possessing the carboxyl-terminus) with a molecular mass of 105 to 130 kD and is resistant to alkali treatment. Immunoelectron microscopy shows platelet APP to be localized to the alpha-granule. Activation of platelets results in a threefold increase in surface APP detectability. In plasma, the 130-kD APP-reactive band is increased in AD. We find that in the gray platelet syndrome, platelets contain reduced amounts of APP, with a corresponding reduction in plasma APP levels, suggesting that platelets are the major source of plasma APP. Our studies also identify an interaction of APP with platelet membranes which differs from that found in the brain, and raise the possibility of a receptor for APP in platelet membranes. Quantitative differences in the amounts of APPMem in platelets compared with brain also indicate regulation of the pathways that determine the cleavage of APP near its transmembrane domain. These pathways are a therapeutic target in AD, and may be easily amenable to investigation in platelets.

The characteristic pathogenic feature of polymyositis (PM) is muscle invasion by T lymphocytes penetrating the basal lamina and displacing the sarcolemma of normal muscle fibers (T cell invasion of endomysial tubes). Active forward movement of these T cells is indicated by cell extensions interdigitating with the muscle fiber surface. Here we describe for the first time high abundance of Alzheimer amyloid protein precursor (APP) in invasive T cells contacting the border of muscle fibers in PM. These are the sites of muscle fiber displacement. The percentage of APP+ T cells at these sites is significantly higher than in other neuromuscular disorders with inflammatory infiltrates suggesting a specific pathogenic function of these cells in PM. By using a new multiparameter immunofluorescence imaging procedure and confocal laser scanning microscopy, we show that APP+ T cells in PM are invasive front cells that penetrate the basal lamina of the endomysial tube and displace the muscle fiber. Mononuclear cells behind the invasive front are negative for APP or show much lower APP levels. Front T cells either express the CD8-CD4+APP+ or CD8+CD4-APP+ phenotypes, or are CD4+CD8+APP+ T cell chimeras. The highest APP concentration is found at the tip of T cell extensions interdigitating with the fiber surface. Although normal by morphological criteria, the same fibers show intense staining for the regeneration marker NCAM. This reactivity is highest at sites contacted by the APP+ T cells. The findings indicate that APP is specifically upregulated in T cells displacing muscle fibers in PM and suggest that NCAM, which may be abnormally regulated in these fibers, is a candidate molecule for interaction with APP.

The deposition of amyloid protein aggregates in brain is the main pathological feature of Alzheimer's disease. Their principal constituent is a peptide termed beta A4, which comprises up to 43 amino acid residues. It is highly insoluble under physiological conditions and aggregates into filaments that form very dense clusters in vivo and in vitro. Based on a beta A4 prototype sequence spanning residues 10 to 42 or 43, we have designed analogues in which hydrophobic amino acid residues in position 17 to 20 were substituted by more hydrophilic residues. Depending on the kind of newly introduced amino acids and their position within the sequence, the substitution of only two residues led to variants exhibiting a broad spectrum of different properties. Common to them was a reduced beta-sheet content after solubilization in water and in the solid state. Some of the variants showed significantly reduced amyloidogenicity: although still forming filaments, they did not aggregate into the highly condensed depositions that are typical for amyloid. In addition, they could be solubilized in 200 mM-NaCl and KCl. When mixed with beta A4 peptides bearing the natural sequence, two of the analogues could inhibit the formation of filaments in vitro. These results demonstrate that a well-preserved hydrophobic core around residues 17 to 20 of beta A4 is crucial for the formation of beta-sheet structure and the amyloid properties of beta A4. The introduction of structural alterations within this region may guide the development of reagents for the therapy of Alzheimer's disease.

The effects of long-term exposure to aluminium on the development of Alzheimer-type neuropathological changes have been studied post-mortem in patients with chronic renal failure who did not have dialysis encephalopathy. Administration of aluminium-containing phosphate binding compounds appears to be a major factor in the accumulation of aluminium in the brain of dialysis patients. The mean serum aluminium concentrations determined during life and brain aluminium concentrations determined post-mortem correlated with both the duration and total amount of aluminium hydroxide administered to these patients. No correlation was found between the presence of bone aluminium and either the mean serum or brain aluminium concentration. Longitudinal monitoring of serum aluminium concentrations may provide a more reliable index than bone biopsy of brain aluminium concentrations in dialysis patients. Dynamic secondary ion mass spectrometry revealed focal accumulations of aluminium associated with cortical pyramidal neurones. The majority of patients also showed immunostaining in pyramidal neurones with an antibody to the N-terminal region of the beta/A4 amyloid precursor protein, while staining was absent in age-matched control cases. One-third of the patients exhibited beta/A4-positive amorphous senile plaques in the cerebral cortex. However, there was no clear correlation between either the presence and intensity of beta/A4 amyloid precursor immunostaining or the presence of senile plaques and the concentration of aluminium in the cerebral cortex. Cortical neurofibrillary tangles were not observed in any of the dialysis patients. These data suggest that it is unlikely that aluminium plays any major role in neurofibrillary tangle formation and that its putative role in senile plaque formation is likely to be only part of a complex cascade of changes.

To clarify the relationship between amyloid formation and amyloid precursor protein (APP), the brain sections from eight patients with Alzheimer's disease (AD) and four with Gerstmann-Sträussler Syndrome (GSS) were investigated immunohistochemically by the double-immunostaining method. In AD, most APP-positive senile plaques belong to classical plaques or primitive plaques, whereas in diffuse plaques, APP-positive neuritic components are rarely observed. The authors documented that anti-APP-labeled degenerative neurites surrounding kuru plaques in all four GSS patients. These kuru plaques were verified by double immunostaining using anti-prion protein and anti-APP. The APP-positive structures in kuru plaques were almost identical with those seen in the classical plaques in AD. The authors concluded that APP-positive degenerative neurites are not an early event in the amyloid formation of senile plaques. It is therefore postulated that depositions of beta/A4 and prion proteins are primary events that may involve the surrounding microenvironment and result in the secondary formation of APP-positive degenerative neurites, not specific to AD.

The deposition of amyloid beta A4 in the brain is a major pathological hallmark of Alzheimer's disease. Amyloid beta A4 is a peptide composed of 42 or 43 amino acid residues. In brain, it appears in the form of highly insoluble, filamentous aggregates. Using synthetic peptides corresponding to the natural beta A4 sequence as well as analog peptides, we demonstrate requirements for filament formation in vitro. We also determine aggregational properties and the secondary structure of beta A4. A comparison of amino-terminally truncated beta A4 peptides identifies a peptide spanning residues 10 to 43 as a prototype for amyloid beta A4. Infrared spectroscopy of beta A4 peptides in the solid state shows that their secondary structure consists of a beta-turn flanked by two strands of antiparallel beta-pleated sheet. Analog peptides containing a disulfide bridge were designed to stabilize different putative beta-turn positions. Limited proteolysis of these analogs allowed a localization of the central beta-turn at residues 26 to 29 of the entire sequence. Purified beta A4 peptides are soluble in water. Size-exclusion chromatography shows that they form dimers that, according to circular dichroism spectroscopy, adopt a beta-sheet conformation. Upon addition of salts, the bulk fraction of peptides precipitates and adopts a beta-sheet structure. Only a small fraction of peptides remains solubilized. They are monomeric and adopt a random coil conformation. This suggests that the formation of aggregates depends upon a hydrophobic effect that leads to intra- and intermolecular interactions between hydrophobic parts of the beta A4 sequence. This model is sustained by the properties of beta A4 analogs in which hydrophobic residues were substituted. These peptides show a markedly increased solubility in salt solutions and have lost the ability to form filaments. In contrast, the substitution of hydrophilic residues leads only to small deviations in the shape of filaments, indicating that hydrophilic residues contribute to the specificity of interactions between beta A4 peptides.

The evolution of senile plaques and the relationships among neuritic elements, extracellular deposits of the beta-amyloid protein (beta/A4), and vascular beta/A4 are poorly understood. Immunocytochemical methods were used to examine fixed-frozen prefrontal cortices of 14 rhesus monkeys (Macaca mulatta) (14 to 37 years of age) for the presence of abnormal fibers/neurites, alpha 1-antichymotrypsin (alpha-ACT), and beta/A4. Age-associated alterations included abnormal fibers/neurites, presence of beta/A4, and association of alpha-ACT with beta/A4 in plaques and blood vessels. Vascular amyloid was present only in the oldest monkeys. The topographic distribution of abnormal fibers/neurites was mapped with acetylcholinesterase (AChE) histochemistry, and deposits of amyloid were visualized with immunocytochemistry for beta/A4. beta/A4 often was associated with neurites, but many neurites lacked demonstrable beta/A4. Thus in aged monkeys, abnormal neurites may provide one type of focus for the accumulation of the amyloid precursor, which is subsequently abnormally processed to form beta/A4. Our data in rhesus monkeys suggest that fiber and neuritic abnormalities increase with age and that they may precede the majority of beta/A4 deposits; the initial stages of neurite formation and parenchymal amyloid deposits may be independent of the appearance of vascular amyloid; and these processes may be synergistic with advanced age.

Any explanation of the causes of Alzheimer's disease and of its unique cerebral pathologic features must take into account the distribution and ultrastructural localization of the pre-A4 amyloid proteins in tissues and organs. The authors have analyzed the expression of the pre-A4 amyloid proteins in several tissues by immunogold electron microscopy and by immunofluorescence. For this purpose, they have used a mouse monoclonal antibody and a guinea pig antiserum raised against two synthetic peptides corresponding to two different sequences common to all the full-length forms of the A4 amyloid precursors. They observed a tissue-specific distribution of the secreted and the transmembrane form of the precursors. The authors could determine that the secreted form is generated in vivo within the cytoplasm. In the salivary glands and in the adenohypophysis, all the immunoreactivity is associated with the process of secretion, whereas in the muscle, a staining pattern compatible with the presence of the pre-A4 amyloid proteins in the sarcoplasmic reticulum has been observed. This difference in the localization may reflect tissue-specific processing pathways and suggests that posttranslational modifications such as proteolytic removal of the transmembrane and cytoplasmic domains contribute to the structural and thus functional diversity of the A4 amyloid precursors.

A mast cell-like cell line (SN-1) was established with the aid of growth factor(s) present in the supernatant of a Con A-stimulated L3T4+ T cell line. In analogy to other mast cell lines, IL 3 was identified as a growth factor for SN-1 cells. In addition, a second lymphokine produced by the T cells synergistically enhanced the IL 3-induced growth. This factor, originally termed mast cell growth enhancing factor (MaGEF), could be separated from IL 2, IL 3, and a CSF-like activity and was purified to homogeneity. The N-terminal amino acid sequence (8 residues) and the functional properties of this lymphokine proved to be identical with those reported for BSF-1 (IL 4). Unless applied at high concentrations, purified MaGEF did not stimulate growth of the SN-1 mast cells in the absence of IL 3. MaGEF was also found to act on two IL 2-dependent T cell lines by inducing significant thymidine incorporation which was suboptimal compared to that induced by IL 2 and which cannot be inhibited by anti-IL 2-antibodies. A panel of cell lines developed from mouse bone marrow with IL 3 or with a combination of IL 3 and MaGEF all reacted to MaGEF in the presence of IL 3 with considerably increased proliferation. It is therefore suggested that one of the physiological functions of MaGEF is to promote the recruitment of T-dependent mast cells.

Human monoclonal antibodies (MoAbs) directed against the blood group Rh(D), (c), and (E) antigens produced by Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines have been used to characterize the Rh components of human red cell membranes and to determine whether the Rh(D), (c), and (E) epitopes are carried by distinct polypeptides. After immunoprecipitation with the anti-Rh(D) antibody and preparative gel electrophoresis, a homogenous preparation of the Rh(D) protein was obtained from two different erythrocyte samples (Blo and D--/D--), which have an increased density of Rh(D) antigen. Both preparations exhibited the same N-terminal sequence (S)-(S)-K-Y-P-R-S-V-R-R-(L)-L-P-L-X-A, indicating that different Rh(D)-positive red cells are carrying a similar Rh(D) protein. Comparative immunoprecipitation studies with the human monoclonal anti-Rh(D), (c), and (E) antibodies have also shown that Rh components from intact and papain-treated erythrocytes have similar apparent mol wt of 30 to 32 Kd and are buried in the lipid bilayer and are not readily available to the proteolytic enzyme. Further investigations by indirect affinity chromatography and one-dimensional peptide mapping of the Rh(D), (c), and (E) molecules immunopurified from a single red cell sample demonstrate that a common Rh haplotype encodes three distinct polypeptide chains carrying the Rh(D), (c), and (E) epitopes, respectively.