Generation of antibodies specific for beta-amyloid by vaccination of patients with Alzheimer disease

To characterize antibodies produced in humans in response to Abeta42 vaccination, we carried out immunohistochemical examinations of the brains of both transgenic mice and human patients with beta-amyloid pathology. We collected sera from patients with Alzheimer disease who received a primary injection of pre-aggregated Abeta42 followed by one booster injection in a placebo-controlled study. Antibodies in immune sera recognized beta-amyloid plaques, diffuse Abeta deposits and vascular beta-amyloid in brain blood vessels. The antibodies did not cross-react with native full-length beta-amyloid precursor protein or its physiological derivatives, including soluble Abeta42. These findings indicate that vaccination of AD patients with Abeta42 induces antibodies that have a high degree of selectivity for the pathogenic target structures. Whether vaccination to produce antibodies against beta-amyloid will halt the cognitive decline in AD will depend upon clinical assessments over time.     

Alzheimer's disease beta-secretase BACE1 is not a neuron-specific enzyme

The brains of Alzheimer's disease (AD) patients are morphologically characterized by neurofibrillar abnormalities and by parenchymal and cerebrovascular deposits of beta-amyloid peptides. The generation of beta-amyloid peptides by proteolytical processing of the amyloid precursor protein (APP) requires the enzymatic activity of the beta-site APP cleaving enzyme 1 (BACE1). The expression of this enzyme has been localized to the brain, in particular to neurons, indicating that neurons are the major source of beta-amyloid peptides in brain. Astrocytes, on the contrary, are known to be important for beta-amyloid clearance and degradation, for providing trophic support to neurons, and for forming a protective barrier between beta-amyloid deposits and neurons. However, under certain conditions related to chronic stress, the role of astrocytes may not be beneficial. Here we present evidence demonstrating that astrocytes are an alternative source of BACE1 and therefore may contribute to beta-amyloid plaque formation. While resting astroyctes in brain do not express BACE1 at detectable levels, cultured astrocytes display BACE1 promoter activity and express BACE1 mRNA and enzymatically active BACE1 protein. Additionally, in animal models of chronic gliosis and in brains of AD patients, there is BACE1 expression in reactive astrocytes. This would suggest that the mechanism for astrocyte activation plays a role in the development of AD and that therapeutic strategies that target astrocyte activation in brain may be beneficial for the treatment of AD. Also, there are differences in responses to chronic versus acute stress, suggesting that one consequence of chronic stress is an incremental shift to different phenotypic cellular states.     

Inhibition of glycosphingolipid biosynthesis reduces secretion of the beta-amyloid precursor protein and amyloid beta-peptide

Alzheimer disease is associated with extracellular deposits of amyloid beta-peptides in the brain. Amyloid beta-peptides are generated by proteolytic processing of the beta-amyloid precursor protein by beta- and gamma-secretases. The cleavage by secretases occurs predominantly in post-Golgi secretory and endocytic compartments and is influenced by cholesterol, indicating a role of the membrane lipid composition in proteolytic processing of the beta-amyloid precursor protein. To analyze the role of glycosphingolipids in these processes we inhibited glycosyl ceramide synthase, which catalyzes the first step in glycosphingolipid biosynthesis. The depletion of glycosphingolipids markedly reduced the secretion of endogenous beta-amyloid precursor protein in different cell types, including human neuroblastoma SH-SY5Y cells. Importantly, secretion of amyloid beta-peptides was also strongly decreased by inhibition of glycosphingolipid biosynthesis. Conversely, the addition of exogenous brain gangliosides to cultured cells reversed these effects. Biochemical and cell biological experiments demonstrate that the pharmacological reduction of cellular glycosphingolipid levels inhibited maturation and cell surface transport of the beta-amyloid precursor protein. In the glycosphingolipid-deficient cell line GM95, cellular levels and maturation of beta-amyloid precursor protein were also significantly reduced as compared with normal B16 cells. Together, these data demonstrate that glycosphingolipids are implicated in the regulation of the subcellular transport of the beta-amyloid precursor protein in the secretory pathway and its proteolytic processing. Thus, enzymes involved in glycosphingolipid metabolism might represent targets to inhibit the production of amyloid beta-peptides.     

Analysis and Quantitation of the β-Amyloid Precursor Protein in the Cerebrospinal Fluid of Alzheimer''s Disease Patients with a Monoclonal Antibody-Based Immunoassay

One of the major clinical findings in Alzheimer's disease (AD) is the formation of deposits of beta-amyloid protein in amyloid plaques, derived from the beta-amyloid precursor protein (beta-APP). To determine the possible use of beta-APP as a diagnostic marker for AD in CSF, a monoclonal antibody-based immunoassay specific for this protein was developed. The assay does not differentiate between beta-APP695 and beta-APP751 forms but does preferentially recognize beta-APP751 complexed with a protease. Of the two sets of CSF samples tested, one set, obtained from living patients, gave a slightly lower level of beta-APP in AD and Parkinson's disease patients relative to controls, whereas the other set, composed of postmortem samples, showed no significant differences between the AD and control groups.     

Targeted control of kinetics of beta-amyloid self-association by surface tension-modifying peptides

Brain tissue from Alzheimer's patients contains extracellular senile plaques composed primarily of deposits of fibrillar aggregates of beta-amyloid peptide. beta-Amyloid aggregation is postulated to be a major factor in the onset of this neurodegenerative disease. Recently proposed is the hypothesis that oligomeric intermediates, rather than fully formed insoluble fibrils, are cytotoxic. Previously, we reported the discovery of peptides that accelerate beta-amyloid aggregation yet inhibit toxicity in vitro, in support of this hypothesis. These peptides contain two domains: a recognition element designed to bind to beta-amyloid and a disrupting element that alters beta-amyloid aggregation kinetics. Here we show that the aggregation rate-enhancing activity of the disrupting element correlates strongly with its ability to increase surface tension of aqueous solutions. Using the Hofmeister series as a guide, we designed a novel peptide with terminal side-chain trimethylammonium groups in the disrupting domain. The derivatized peptide greatly increased solvent surface tension and accelerated beta-amyloid aggregation kinetics by severalfold. Equivalent increases in surface tension in the absence of a recognition domain had no effect on beta-amyloid aggregation. These results suggest a novel strategy for targeting localized changes in interfacial energy to specific proteins, as a way to selectively alter protein folding, stability, and aggregation.     

Kinetics of aggregation of synthetic beta-amyloid peptide.

beta-Amyloid peptide is the major protein component of senile plaques and cerebrovascular amyloid deposits in patients with Alzheimer's disease. The peptide deposits extracellularly in the form of amyloid fibrils, in a cross-beta conformation. beta-amyloid peptide is a 39- to 43-residue segment of a normal membrane precursor protein. In this work, a peptide homologous to the first 40 amino acids of beta-amyloid peptide, beta(1-40), was synthesized and characterized. beta(1-40) exhibited a sharp change in solubility near physiological pH and gel formation at concentrations of 3 mg/ml or greater. Circular dichroism indicated that beta(1-40) contained approximately two-thirds beta-structure, but no alpha-helical character. Quasi-elastic and classical light scattering measurements showed that beta(1-40) aggregated end-to-end in solution, reaching average molecular weights greater than 4 x 10(6) after 13 days. The aggregates were best modeled as rigid rods of 5 nm diameter, similar to the diameter of amyloid fibrils purified from plaques. A mathematical model based on diffusion-limited aggregation was developed to describe the kinetics of aggregation.     

Transition metal-mediated glycoxidation accelerates cross-linking of beta-amyloid peptide.

beta-Amyloid deposits, hallmarks of Alzheimer's disease, contain both sugar-derived 'advanced glycation end products' (AGEs) and copper and iron ions. Our in vitro experiments using synthetic beta-amyloid peptide and glucose or fructose show that formation of covalently cross-linked high-molecular-mass beta-amyloid peptide oligomers is accelerated by micromolar amounts of copper (Cu+, Cu2+) and iron (Fe2+, Fe3+) ions. Formation of these covalent AGE cross-links can be inhibited by capping agents of amino groups, redox-inactive metal chelators and antioxidants, suggesting that these drugs may be able to slow down the formation of insoluble beta-amyloid deposits in vivo and possibly the progression of Alzheimer's disease.     

In situ characterization of beta-amyloid in Alzheimer''s diseased tissue by synchrotron Fourier transform infrared microspectroscopy.

We report the first evidence of the structure of beta-amyloid protein as it exists in situ within a slice of human Alzheimer's diseased brain tissue. Using a Fourier transform infrared microspectroscopic technique, areas of interest can be selected for spectral measurements with regions of potential contamination masked. In so doing, it is possible to obtain infrared spectra only of beta-amyloid and not the surrounding grey matter within which it lies. However, to obtain spectra of high-quality signal-to-noise ratio using a conventional infrared source, we were limited to aperture sizes between 24 microns x 24 microns to 50 microns x 50 microns. Markedly improved high-quality spectra were acquired with infrared radiation provided by a synchrotron light source (National Synchrotron Light Source, Brookhaven National Laboratories), using aperture sizes as small as 12 microns x 12 microns. This allowed spectroscopic mapping of brain tissue regions containing amyloid. We observe that in situ protein of grey matter exist predominantly in an alpha-helical and/or unordered conformation, whereas within amyloid deposits a beta-sheet structure predominates. The hydrogen bonding strength of the beta-structure found in situ is different from that reported in the literature for isolated/chemically synthesized beta-amyloid peptides.     

Overexpression of the neuritotrophic cytokine S100beta precedes the appearance of neuritic beta-amyloid plaques in APPV717F mice

Homozygous APPV717F transgenic mice overexpress a human beta-amyloid precursor protein (betaAPP) minigene encoding a familial Alzheimer's disease mutation. These mice develop Alzheimer-type neuritic beta-amyloid plaques surrounded by astrocytes. S100beta is an astrocyte-derived cytokine that promotes neurite growth and promotes excessive expression of betaAPP. S100beta overexpression in Alzheimer's disease correlates with the proliferation of betaAPP-immunoreactive neurites in beta-amyloid plaques. We found age-related increases in tissue levels of both betaAPP and S100beta mRNA in transgenic mice. Neuronal betaAPP overexpression was found in cell somas in young mice, whereas older mice showed betaAPP overexpression in dystrophic neurites in plaques. These age-related changes were accompanied by progressive increases in S100beta expression, as determined by S100beta load (percent immunoreactive area). These increases were evident as early as 1 and 2 months of age, months before the appearance of beta-amyloid deposits in these mice. Such precocious astrocyte activation and S100beta overexpression are similar to our earlier findings in Down's syndrome. Accelerated age-related overexpression of S100beta may interact with age-associated overexpression of mutant betaAPP in transgenic mice to promote development of Alzheimer-like neuropathological changes.     

Identification and characterization of C-terminal fragments of the beta-amyloid precursor produced in cell culture.

The mechanism of amyloid formation in Alzheimer's disease is unknown but appears to involve proteolytic processing of the amyloidogenic peptide from a larger precursor. When the C-terminus containing the amyloid-forming and cytoplasmic domains of the precursor was recombinantly expressed in cultured mammalian cells, a 16 kd protein accumulated which had a tendency to aggregate and form deposits. These deposits had physical characteristics resembling those of preamyloid. Recombinant expression of the full-length precursor was found to produce a similar, cell-associated 16 kd C-terminal fragment as well as a 12 kd fragment, neither of which formed detectable aggregates suggesting efficient catabolism of these fragments. Identification of these two naturally occurring metabolic products of the beta-amyloid precursor provides a system permitting the characterization of the proteolytic processing events of the amyloid precursor protein.     

Neurofibrillary tangles and beta-amyloid deposits in Alzheimer's disease.

Alzheimer's disease is characterized by the presence of abundant neurofibrillary tangles and beta-amyloid deposits in neocortex, hippocampus and amygdala. The major protein components of tangles and plaques have recently been identified. These findings, briefly reviewed here, will allow researchers to design investigations that will lead to an understanding of the pathogenesis of the disease and to the development of new therapeutic approaches that may result in an effective treatment.     

Chronic nicotine treatment reduces beta-amyloidosis in the brain of a mouse model of Alzheimer's disease (APPsw)

Alzheimer's disease neuropathology is characterised by beta-amyloid plaques and neurofibrillary tangles. Inhibition of beta-amyloid accumulation may be essential for effective therapy in Alzheimer's disease. In this study we have treated transgenic mice carrying the Swedish mutation of human amyloid precursor protein [Tg(Hu.APP695.K670N-M671L)2576], which develop brain beta-amyloid deposits, with nicotine in drinking fluid (200 microg/mL) from 9-14.5 months of age (5.5 months). A significant reduction in amyloid beta peptide 1-42 positive plaques by more than 80% (p < 0.03) was observed in the brains of nicotine treated compared to sucrose treated transgenic mice. In addition, there was a selective reduction in extractable amyloid beta peptides in nicotine treated mice; cortical insoluble 1-40 and 1-42 peptide levels were lower by 48 and 60%, respectively (p < 0.005), whilst there was no significant change in soluble 1-40 or 1-42 levels. The expression of glial fibrillary acidic protein was not affected by nicotine treatment. These results indicate that nicotine may effectively reduce amyloid beta peptide aggregation in brain and that nicotinic drug treatment may be a novel protective therapy in Alzheimer's disease.     

Glycosaminoglycans and beta-amyloid,prion and tau peptides in neurodegenerative diseases

Protein aggregation into dense filamentous inclusions is a characteristic feature of many etiologically diverse neurodegenerative disorders including Alzheimer's disease (AD), spongiform encephalopathies, and tauopathies. Thus, beta-amyloid peptide (Abeta) accumulates within senile amyloid plaques in AD, protease-resistant prion protein constitutes the amyloid deposits in spongiform encephalopathies and tau protein gives rise to neurofibrillary tangles (NFT) both in AD and in tauopathies. Curiously, these abnormal protein inclusions contain, in addition to their major peptide components, some associated sulfated glycosaminoglycans (sGAG). Here we discuss the proposal that the binding of sGAG to aggregate-forming peptides may modify the pathogenic process depending on their subcellular localization.     

Alzheimer's disease therapeutics: new approaches to an ageing problem

Abnormal proteinaceous deposits are found in the brain of patients with many different neurodegenerative diseases. In many of these diseases, the production of the deposits is probably associated with disease pathogenesis. In Alzheimer's disease (AD), the amyloid protein (A beta), is produced by the action of enzymes known as secretases, which cleave the beta-amyloid protein precursor. A beta is secreted from cells in the brain, after which it oligomerizes and is deposited in the extracellular compartment of the brain to form amyloid plaques and amyloid angiopathy. Targeting the production of A beta and its aggregation is now a key strategy in the development of novel therapeutic agents for the treatment of AD. This review examines the potential of immunization strategies, cholesterol-lowering drugs, protease inhibitors and nicotinic drugs for the treatment of AD.     

Glycosaminoglycans inhibit neurodegenerative effects of serum amyloid P component in vitro

Serum amyloid P component, a member of pentraxin serum protein family, has been suggested to contribute to the progression of neurodegeneration including Alzheimer's disease by binding to beta-amyloid fibrils leading to an increased stability of the deposits against proteolytic degradation and by inducing neuronal apoptosis. Here, we show that glycosaminoglycans inhibit both the serum amyloid P component-beta-amyloid interaction and the neurotoxic effect of serum amyloid P component. These effects correlate with the structure of glycosaminoglycans and show different structure-activity relationship in the case of the two different effects. While the efficacy of the inhibition on the serum amyloid P component-induced cell death increases with the uronic acid content, the inhibitory activity on the serum amyloid P component-beta-amyloid interaction decreases with the increasing uronic acid content of the glycosaminoglycans. The inhibitory effect of glycosaminoglycans on the interaction between the first component of the complement cascade (C1q) and beta-amyloid shows a similar structure-activity relationship as on the serum amyloid P component-beta-amyloid interaction. This suggests that glycosaminoglycans interfere with the binding site on beta-amyloid for serum amyloid P component and C1q. The functional consequence of this binding has been demonstrated by heparin which promotes the proteolysis of beta-amyloid in vitro in the presence of serum amyloid P component. Our results suggest that glycosaminoglycans might have therapeutical potential on the neurodegeneration reducing its progress.     

Endogenous proteins controlling amyloid beta-peptide polymerization. Possible implications for beta-amyloid formation in the central nervous system and in peripheral tissues.

We report that certain plasma proteins, at physiological concentrations, are potent inhibitors of amyloid beta-peptide (Abeta) polymerization. These proteins are also present in cerebrospinal fluid, but at low concentrations having little or no effect on Abeta. Thirteen proteins representing more than 90% of the protein content in plasma and cerebrospinal fluid were studied. Quantitatively, albumin was the most important protein, representing 60% of the total amyloid inhibitory activity, followed by alpha1-antitrypsin and immunoglobulins A and G. Albumin suppressed amyloid formation by binding to the oligomeric or polymeric Abeta, blocking a further addition of peptide. This effect was also observed when the incorporation of labeled Abeta into genuine beta-amyloid in tissue section was studied. The Abeta and the anti-diabetic drug tolbutamide apparently bind to the same site on albumin. Tolbutamide displaces Abeta from albumin, increasing its free concentration and enhancing amyloid formation. The present results suggest that several endogenous proteins are negative regulators of amyloid formation. Plasma contains at least 300 times more amyloid inhibitory activity than cerebrospinal fluid. These findings may provide one explanation as to why beta-amyloid deposits are not found in peripheral tissues but are only found in the central nervous system. Moreover, the data suggest that some drugs that display an affinity for albumin may enhance beta-amyloid formation and promote the development of Alzheimer's disease.     

CD36 signals to the actin cytoskeleton and regulates microglial migration via a p130Cas complex

The pattern recognition receptor CD36 initiates a signaling cascade that promotes microglial activation and recruitment to beta-amyloid deposits in the brain. In the present study we identify the focal adhesion-associated proteins p130Cas, Pyk2, and paxillin as novel members of the tyrosine kinase signaling pathway downstream of CD36 and show that assembly of this complex is essential for microglial migration. In primary microglia and macrophages exposed to beta-amyloid, the scaffolding protein p130Cas is rapidly tyrosine-phosphorylated and co-localizes with CD36 to membrane ruffles contemporaneous with F-actin polymerization. These beta-amyloid-stimulated events are not detected in CD36 null cells and are dependent on CD36 activation of Src family tyrosine kinases. Fyn, a Src kinase known to interact with CD36, co-precipitates with p130Cas and is an essential upstream intermediate in the signaling pathways leading to phosphorylation of the p130Cas substrate domain. Furthermore, the p130Cas-interacting kinase Pyk2 and the cytoskeletal adapter protein paxillin also demonstrate CD36-dependent phosphorylation, identifying these focal adhesion molecules as additional members of this beta-amyloid signaling cascade. Disruption of this p130Cas complex by small interfering RNA silencing inhibits p44/42 mitogen-activated protein kinase phosphorylation and microglial migration, illustrating the importance of this pathway in microglial activation and recruitment. Together, these data are the first to identify the signaling cascade that directly links CD36 to the actin cytoskeleton and, thus, implicates it in diverse processes such as cellular migration, adhesion, and phagocytosis.     

Alzheimer's and Parkinson's disease--overlapping or synergistic pathologies?

Alzheimer's disease (AD) and Parkinson's disease (PD) are the two most common neurodegenerative disorders in humans. They are characterized by insoluble protein deposits; beta-amyloid plaques and tau-containing neurofibrillary lesions in AD, and alpha-synuclein-containing Lewy bodies in PD. As a significant percentage of patients have clinical and pathological features of both diseases, the patho-cascades of the two diseases might overlap. For the first time, new animal models that express multiple transgenes provide the tools to dissect the pathogenic pathways and to differentiate between additive and synergistic effects.     

A(beta) generation in autophagic vacuoles

Alzheimer's disease (AD) is the most common form of dementia among older people. It is characterized by the extracellular accumulation of beta-amyloid (Abeta) deposits called senile or neuritic plaques. Abeta is generated by the proteolytic cleavage of Abeta precursor protein (APP) by beta and gamma-secretases localized in the secretory and endocytic compartments. In this issue, Yu et al. (on p. 87) report a novel mechanism for the generation of Abeta peptides, which takes place in autophagic vacuoles (AVs) that accumulate in AD brains.     

A demonstration of the advantages of immunostaining in the quantification of amyloid plaque deposits

Extracellular amyloid deposits are a feature of both Alzheimer type dementia and the 'normal' aging process. Quantification of amyloid plaque deposits may well be useful in distinguishing between the senescent changes associated with 'normal' aging and the pathological processes underlying dementia. To determine the most reliable and reproducible method for visualisation of the amyloid we have compared conventional silver staining techniques with beta-amyloid immunocytochemistry on a large sample of post-mortem brain tissue from both demented (n = 15, age range 60-87) and non-demented (n = 65, age range 14-99) patients. The degree of amyloid deposition was rated on a four point scale and ratings for the two techniques were significantly correlated (P less than 0.01). However, the immunocytochemical approach has a number of distinct advantages for quantification. The antibody to beta-amyloid is highly specific and does not stain neurofibrillary tangles or background features, it is considerably more sensitive than silver staining in highlighting diffuse amyloid deposits and, perhaps most importantly, it produces high contrast staining which allows easier image digitisation and subsequent computer image analysis.