Selective binding of soluble Abeta1-40 and Abeta1-42 to a subset of senile plaques.

Alzheimer's disease is characterized by the progressive accumulation of amyloid-beta protein (Abeta) in senile plaques and cerebral amyloid angiopathy. It is not known whether the plaque growth is a continuous and homogeneous process or whether some plaques have a more rapid evolution. As plaques grow by the deposition of Abeta, we used an in situ binding technique to analyze the deposition of fluorescein-conjugated and biotinylated Abeta1 40 and Abeta1-42 in cryosections of brains from Alzheimer's disease patients. Only a subset of senile plaques but all cerebrovascular Abeta deposits were labeled by both Abeta1-40 and Abeta1-42. Striking differences in binding were observed among adjacent plaques. Quantitative analysis showed that on average 60% of all plaques were labeled with Abeta1-42 and 31% of all plaques were labeled with Abeta1-40 (n=7; P<0.001). Confocal laser scanning microscopy of double-labeled sections revealed that the newly deposited Abeta was only partially co-localized to pre-existing Abeta and apolipoprotein E and was not co-localized to heparan sulfate proteoglycan. Abeta binding was preserved after glycolytic pretreatment with periodic acid. Our results suggest that at a given time point only a subset of active senile plaques accumulate A(beta) and that plaque growth may be conditioned by the presence of other distinct plaque components different from Abeta, apolipoprotein E or heparan sulfate proteoglycan.     

Uptake of Abeta 1-40- and Abeta 1-42-coated yeast by microglial cells: a role for LRP

Artificial diffuse and amyloid core of neuritic plaques [beta-amyloid peptide (Abeta) deposits] could be prepared using heat-killed yeast particles opsonized with Abeta 1-40 or Abeta 1-42 peptides. Interaction and fate of these artificial deposits with microglial cells could be followed using a method of staining that allows discrimination of adherent and internalized, heat-killed yeast particles. Using this system, it was possible to show that nonfibrillar or fibrillar (f)Abeta peptides, formed in solution upon heating (aggregates), could not impair the internalization of heat-killed yeast particles opsonized with fAbeta 1-40 or fAbeta 1-42. This indicated that depending on their physical state, Abeta peptide(s) do not recognize the same receptors and probably do not follow the same internalization pathway. Using competitive ligands of class A scavenger receptors (SR-A) or low-density lipoprotein-related receptor protein (LRP), it has been shown that SR-A were not involved in the recognition of amyloid peptide deposits, whereas LRP specifically recognized deposits of fAbeta 1-42 (but not fAbeta 1-40) and mediated their phagocytosis.     

Abeta(1-42) stimulated T cells express P-PKC-delta and P-PKC-zeta in Alzheimer disease

The protein kinase C (PKC) family of enzymes is a regulator of transmembrane signal transduction, and involvement of some PKC isoforms in T-cell activation has been demonstrated. Nevertheless, very little is known about their involvement in the Amyloid beta (Abeta)-dependent molecular signals in the T lymphocytes of Alzheimer disease (AD) patients. Therefore, the aim of this study was to investigate the involvement of PKC-alpha, PKC-delta and PKC-zeta expression and activity in the signaling machinery activated in Abeta-reactive T cells, in adult healthy individuals, elderly healthy subjects, and from patients with AD. The results show that in peripheral T-cells from early AD patients, Abeta(1-42) produced a distinct subpopulation highly expressing P-PKC-delta, while in severe AD patients the same treatment induced two distinct P-PKC-delta and P-PKC-zeta T-cell subpopulations. Such subpopulations were not noticeable following CD3/CD28 treatment of the same samples or after treatment of peripheral T cells from healthy adult or elderly subjects with Abeta(1-42) or with CD3/CD28. We believe that these findings may be of help in possible attempts to develop further diagnostic strategies useful for the characterization of AD.     

Immune responses against Abeta1-42 in HLA class II transgenic mice: implications for Abeta1-42 immune-mediated therapies

We have investigated whether polymorphic differences in the major histocompatibility complex (MHC) class II molecules influence humoral and cellular immune responses against Abeta1-42. To analyze the effects of mouse MHC class II and tolerance effects of overexpression of human APP in mice, we immunized Tg2576 and non-transgenic littermates bred into two different MHC backgrounds with Abeta1-42 and compared both B and T cell responses. We found that in the presence of the mouse C57BL/6 background, both B and T cell responses against Abeta1-42 were significantly suppressed. To directly test the contribution of human MHC class II, we immunized various human HLA class II transgenic (TG) mice with Abeta1-42 and analyzed anti-Abeta immune responses. HLA-DR3 and HLA-DQ8 TG mice generated modest B and T cell responses against Abeta1-42. The presence of HLA-DR3/DQ8 in double TG mice enhanced the overall immune response against Abeta1-42. In contrast, HLA-DR4 TG mice mounted strong T cell responses but failed to generate high titer antibody responses against Abeta1-42, whereas, the HLA-DQ6 TG mice were not able to mount significant B or T cell responses against Abeta1-42. These studies in mice suggest that the presence of certain MHC class II molecules or combinations of class II molecules can potentially influence the overall immune response against Abeta1-42.     

Proteomic identification of proteins specifically oxidized in Caenorhabditis elegans expressing human Abeta(1-42): implications for Alzheimer's disease

Protein oxidation has been shown to lead to loss of protein function, increased protein aggregation, decreased protein turnover, decreased membrane fluidity, altered cellular redox poteintial, loss of Ca2+ homeostaisis, and cell death. There is increasing evidence that protein oxidation is involved in the pathogenesis of Alzheimer's disease and amyloid beta-peptide (1-42) has been implicated as a mediator of oxidative stress in AD. However, the specific implications of the oxidation induced by Abeta(1-42) on the neurodegeneration evident in AD are unknown. In this study, we used proteomic techniques to identify specific targets of oxidation in transgenic Caenorhabditis elegans (C. elegans) expressing human Abeta(1-42). We identified 16 oxidized proteins involved in energy metabolism, proteasome function, and scavenging of oxidants that are more oxidized compared to control lines. These results are discussed with reference to Alzheimer's disease.     

Association of CSF apolipoprotein E, Abeta42 and cognition in Alzheimer's disease.

A significant association between CSF Abeta42 and cognition in patients with Alzheimer's disease (AD) homozygous for the epsilon3 allele of the apolipoprotein E (apoE) has been described. In this study we extended our observations on apoE, as another plaque component, and investigated the association between CSF apoE concentrations and cognitive performance after stratification for the apoE genotype in 62 patients with AD, 19 other forms of dementia and 18 controls. CSF Abeta42 and apoE concentrations were significantly and positively associated with Mini Mental State Examination (MMSE) score in AD (Abeta42: r = 0.332; P = 0.026; apoE: r = 0.386; P = 0.006). For Abeta42 this association was exclusively present in epsilon3 homozygotes (r = 0.44; P = 0.014), whereas apoE was correlated with MMSE in epsilon4 hetero- or homozygotes subjects (epsilon4/epsilonX: r = 0.638; P = 0.004: epsilon4/epsilon4; r = 0.812; P = 0.05). No association was observed between CSF concentrations of Abeta42 and apoE. The significant relationship between MMSE and CSF Abeta42 in epsilon3 homozygotes and apoE in epsilon4 hetero- and homozygotes respectively may suggest that both proteins may be associated independently from each other with cognitive decline.     

Autoimmunity in Alzheimer's disease: increased levels of circulating IgGs binding Abeta and RAGE peptides

Plasma samples derived from 33 Alzheimer's disease (AD) and 42 control participants were subjected to several steps to purify specific anti-(amyloid)Abeta IgGs. Affinity-purified IgGs binding the peptide Abeta1-42, a neurotoxic sequence derived from the trans-membrane amyloid precursor protein, exhibited nearly four-fold higher titers in AD patients compared with their control non-AD cohort. Affinity-purified IgGs binding a fragment of the receptor for advanced glycation end products (RAGE) likewise were increased nearly three-fold in AD individuals. Abeta and RAGE IgG titers were negatively correlated with cognitive status, i.e. the more cognitively impaired individuals tended to exhibit higher IgG titers. Abeta IgG titers were negatively correlated with age in the control group, but not with the AD group. Levels of circulating AB- and RAGE-like proteins were not different between AD and control participants, nor was there a relationship between individual IgG titers and the respective Abeta- and RAGE-like proteins. Freshly prepared leukocyte preparations were subjected to flow cytometric analysis. AD individuals exhibited significantly increased populations of cells expressing binding sites for monoclonal antibodies directed against Abeta (5.5-fold), betaAPP (3.5-fold), and RAGE (2.6-fold) relative to the control group. These findings confirm the presence of circulating IgGs specifically directed at proteins implicated in immunological processes linked to AD. The close relationship between titers for Abeta and RAGE IgGs suggests the possibility that the antibodies are being produced in response to a common mechanism or protein complex (with the respective epitopes) linked to the disease.     

C4b-binding protein (C4BP) activates B cells through the CD40 receptor

We demonstrate that the alpha chain of human C4b binding protein (C4BP) binds directly to CD40 on human B cells at a site that differs from that used by CD40 ligand. C4BP induces proliferation, upregulation of CD54 and CD86 expression, and IL4-dependent IgE isotype switching in normal B cells but not in B cells from patients with CD40 or IKKgamma/NEMO deficiencies. Furthermore, C4BP colocalized with B cells in the germinal centers of human tonsils. These observations suggest that C4BP is an activating ligand for CD40 and establish a novel interface between complement and B cell activation.     

Role of the C3b-binding site on C4b-binding protein in regulating classical pathway C5 convertase

A high affinity C5 convertase is generated when a C3 convertase deposits additional C3b molecules on and around itself thereby switching the substrate specificity of C3 convertase from C3 to C5. In the present study the role of the additional C3b molecules in influencing the regulation of classical pathway C5 convertase by C4b-binding protein (C4BP) was examined and compared to its precursor, the C3 convertase. Determination of IC(50) for inhibiting formation of the high affinity C5 convertase and for enhancing its decay (72 and 20 nM) were found to be similar to those obtained for the surface-bound C3 convertase (35 and 11 nM). No difference was observed in the cofactor activity of C4BP for surface-bound C4b alone or when in complex with C3b. Analysis of binding interactions between C4BP and EAC1,C4b cells revealed an average apparent dissociation constant (12 nM) similar to that obtained with EAC1,C4b cells with C3b on them (11 nM). Increasing the C4b or C3b density on the cell surface did not alter the affinity of C4BP. The data suggest that C4BP regulates the C5 convertase by mechanisms similar to those observed for the C3 convertase. Since the IC(50) for inhibiting formation of the soluble C3 convertase (5 nM) is 50-80-fold below the normal serum concentration of C4BP (250-400 nM), C4BP in blood effectively prevents formation of classical pathway C3 convertase in the fluid phase. Although deposition of additional C3b molecules is necessary to convert a C3 convertase to a high affinity C5 convertase, the additional C3b molecules play no role in the regulation of C5 convertase by C4BP.     

Decreased phosphatidylethanolamine binding protein expression correlates with Abeta accumulation in the Tg2576 mouse model of Alzheimer's disease

Phosphatidylethanolamine binding protein (PEBP) is a multifunctional protein, with proposed roles as the precursor protein of hippocampal cholinergic neurostimulating peptide (HCNP), and as the Raf kinase inhibitor protein (RKIP). Previous studies have demonstrated a decrease in PEBP mRNA in CA1 region of AD hippocampus. The current study demonstrates that PEBP is decreased in the hippocampus of 11 month Tg2576 mice, in the absence of change in mRNA levels compared to non-transgenic littermates. The level of PEBP in transgenic mouse hippocampus significantly decreases at 11 months (a time point when Abeta begins accumulating) and 15 months (when Abeta plaques have formed). There was a significant correlation between decreased PEBP expression and accumulation of Abeta. Immunohistochemical studies on Tg2576 and AD brain sections demonstrate that PEBP immunoreactivities are present at the periphery of dense multicore Abeta plaques, and in selective astrocytes, primarily surrounding plaques. These findings suggest that PEBP expression may be influenced by accumulation of Abeta. Down-regulation of PEBP may result in lower levels of HCNP or altered coordination of signal transduction pathways that may contribute to neuronal dysfunction and pathogenesis in AD.     

Heptose I glycan substitutions on Neisseria gonorrhoeae lipooligosaccharide influence C4b-binding protein binding and serum resistance

Lipooligosaccharide (LOS) heptose (Hep) glycan substitutions influence gonococcal serum resistance. Several gonococcal strains bind the classical complement pathway inhibitor, C4b-binding protein (C4BP), via their porin (Por) molecule to escape complement-dependent killing by normal human serum (NHS). We show that the proximal glucose (Glc) on HepI is required for C4BP binding to Por1B-bearing gonococcal strains MS11 and 1291 but not to FA19 (Por1A). The presence of only the proximal Glc on HepI (lgtE mutant) permitted maximal C4BP binding to MS11 but not to 1291. Replacing 1291 lgtE Por with MS11 Por increased C4BP binding to levels that paralleled MS11 lgtE, suggesting that replacement of the Por1B molecule dictated the effects of HepI glycans on C4BP binding. The remainder of the strain background did not affect C4BP binding; replacing the Por of strain F62 with MS11 Por (F62 PorMS11) and truncating HepI mirrored the findings in the MS11 background. C4BP binding correlated with resistance to killing by NHS in most instances. F62 PorMS11 and its lgtE mutant were sensitive to NHS despite binding C4BP, secondary to kinetically overwhelming classical pathway activation and possibly increased alternative pathway activation (measured by factor Bb binding) by the F62 background. FA19 lgtF (HepI unsubstituted) resisted killing by only 10% NHS, not 50% NHS, despite binding levels of C4BP similar to those of FA19 and FA19 lgtE (both resistant to 50% serum), suggesting a role for the proximal Glc in serum resistance independently of C4BP binding. This study provides mechanistic insights into how HepI LOS substitutions affect the serum resistance of N. gonorrhoeae.     

Evaluation of plasma Abeta(40) and Abeta(42) as predictors of conversion to Alzheimer's disease in patients with mild cognitive impairment

Numerous studies have shown a marked decrease of beta-amyloid(42) (Abeta(42)) in the cerebrospinal fluid (CSF) of patients with incipient Alzheimer's disease (AD). However, studies on Abeta in plasma are contradictory, and show very marginal differences between patients and controls. Here, we analyzed plasma samples using a new multiplex immunoassay for simultaneous analysis of Abeta(1-40), Abeta(n-40), Abeta(1-42), and Abeta(n-42). The plasma samples were obtained at baseline from two independent cohorts of patients with mild cognitive impairment (MCI) and age-matched controls. In the first cohort, 41% of the 117 MCI cases converted to AD during a clinical follow-up period of 4-7 years. In the second cohort, 14% of the 110 MCI subjects developed AD during a clinical follow-up period of 2-4 years. None of the plasma Abeta isoforms differed between MCI patients that subsequently developed AD and healthy controls or stable MCI patients. The Cox proportional hazards model did not reveal any differences in the probability of progression from MCI to AD related to plasma Abeta levels. In contrast, low levels of Abeta(1-42) in CSF were strongly associated with increased risk of future AD. The absence of a change in plasma Abeta in incipient AD, despite the marked change in CSF, may be explained by the lack of a correlation between the levels of Abeta(1-42) in CSF and plasma. In conclusion, the results show that CSF biomarkers are better predictors of progression to AD than plasma Abeta isoforms.     

Interest of CSF beta-amyloid1-42 and t-tau protein level determinations for the diagnosis of Alzheimer's disease

Early diagnosis of Alzheimer's disease remains a tactful poser. In order to clarify the importance of beta amyloid protein dosage (Abeta1-42) and protein tau (t-tau) in such pathology, we have rigorously studied three well recruited populations that match in age: healthy controls (n = 32), Alzheimer patients (n = 87) and non Alzheimer dementia (n = 31) patients. The combination of Abeta1-42 and t-tau at baseline yielded a sensitivity of 85.29 % for detection of Alzheimer's disease and the specificity was by 96.77 % to differentiate controls. So the combination of these tow markers helps in the diagnosis of Alzheimer because of the high specificity and sensibility of this method.     

CSF beta-amyloid 1-42 and tau in Tunisian patients with Alzheimer's disease: the effect of APOE epsilon4 allele

Alzheimer's disease (AD) is the leading cause of dementia. Currently, no definitive diagnostic test for AD exists. An accurate, convenient and objective test to detect AD is urgently needed for efficient drug development and effective clinical use of emerging therapies. The aim of the present work is to investigate the usefulness of cerebrospinal fluid (CSF) beta-amyloid protein (Abeta1-42) and total tau protein (t-tau) analyses in the diagnosis of AD and whether apolipoprotein E (ApoE) epsilon4 allele is a factor for AD affecting Tunisian people. Abeta1-42 and t-tau levels were measured in CSF from AD patients (n=73), non-Alzheimer dementia (nAD, n=35) and healthy controls (HC, n=38) by sandwich enzyme-linked immunosorbent assay. Abeta1-42 levels were decreased and t-tau increased in AD patients. The combination of Abeta1-42 and t-tau at baseline yielded a sensitivity of 87.4% for detection of AD. The specificities were 97.3% for controls and 82.7% for other dementia. The ApoE epsilon4 allele frequency (29.5%) was significantly higher in the AD patients than in the nAD patients (17.1%) or in the control groups (9.5%). AD patients carrying ApoE epsilon4 allele had lower Abeta1-42 (p<0.001) levels than those without a epsilon4 allele. The combination of t-tau and Abeta1-42 is a robust and reliable assay that may be useful in discriminating cases at risk for AD such as ApoE epsilon4 allele carriers from nAD patients or from age-matched control subjects.     

CCP1-4 of the C4b-binding protein alpha-chain are required for factor I mediated cleavage of complement factor C3b

C4b-binding protein (C4BP) is a potent regulator of the complement system because it strongly inhibits the classical pathway of complement. Furthermore, C4BP serves as a cofactor to factor I (FI) in the cleavage of fluid phase C3b and can, therefore, influence the alternative pathway of complement. The major form of C4BP in plasma consists of seven identical alpha-chains and one beta-chain. Both types of subunits are composed of complement control protein (CCP) domains, eight such domains make up one alpha-chain. To elucidate the structural requirements for the interaction between C3b and the alpha-chain, nineteen recombinant C4BP variants were used: six truncated monomeric variants, nine polymeric variants in which individual CCPs were deleted, and finally four variants in which double alanine residues were introduced between CCPs. We found that C4BP requires all four N-terminal CCPs of the alpha-chain, with CCP2 and 3 being the most important, to act as a cofactor in the cleavage of C3b. Also, a cluster of positively charged amino acids on the interface between CCP1 and 2 is involved in the binding. Compared to the interaction with C4b, we conclude that binding of C3b to C4BP requires larger molecular surface on C4BP. We found that C4BP was able to act as cofactor in degradation of surface bound C3b and to accelerate decay of alternative C3-convertase. However, in both cases 1,000-fold molar excess of C4BP over factor H (FH), well known inhibitor of the alternative pathway, was required to obtain the same effect.     

Functions of human complement inhibitor C4b-binding protein in relation to its structure

Considering the destructive potential of the complement cascade, it is no surprise that there are several complement inhibitors present in blood and expressed on virtually all cells of the body to protect self tissue. C4b-binding protein (C4BP) is a potent soluble inhibitor of the classical and lectin pathways of complement. This large (500 kDa) plasma glycoprotein consists of seven identical 75 kDa alpha-chains and a unique 40 kDa beta-chain that are held together by disulphide bridges. Both types of subunit are almost exclusively composed of complement control protein (CCP) domains. In recent years, detailed studies of structure-function relationships have yielded new understanding of the interactions between C4BP and the activated complement factors C4b and C3b, heparin, and vitamin K-dependent anticoagulant protein S. This review describes the localization of binding sites for a number of C4BP ligands in relation to well-established and novel functions of C4BP such as complement inhibition, protection of apoptotic cells from complement, CD40-dependent stimulation of B cells, and the contribution of a number of human pathogens to pathogenesis.     

Calcyclin binding protein and Siah-1 interacting protein in Alzheimer's disease pathology: neuronal localization and possible function

The calcyclin binding protein and Siah-1 interacting protein (CacyBP/SIP) protein was shown to play a role in the organization of microtubules. In this work we have examined the neuronal distribution and possible function of CacyBP/SIP in cytoskeletal pathophysiology. We have used brain tissue from Alzheimer's disease (AD) patients and from transgenic mice modeling 2 different pathologies characteristic for AD: amyloid and tau. In the brain from AD patients, CacyBP/SIP was found to be almost exclusively present in neuronal somata, and in control patients it was seen in the somata and neuronal processes. In mice doubly transgenic for amyloid precursor protein and presenilin 1 there was no difference in CacyBP/SIP neuronal localization in comparison with the nontransgenic animals. By contrast in tau transgenic mice, localization of CacyBP/SIP was similar to that observed for AD patients. To find the relation between CacyBP/SIP and tau we examined dephosphorylation of tau by CacyBP/SIP. We found that indeed it exhibited phosphatase activity toward tau. Altogether, our results suggest that CacyBP/SIP might play a role in AD pathology.     

Protective effects of Abeta-derived tripeptide, Abeta(32-34), on Abeta(1-42)-induced phosphatidylinositol 4-kinase inhibition and neurotoxicity

We previously reported that the neurotoxicity of pathophysiological concentrations of amyloid beta proteins (Abetas, 0.1-10nM) as assessed by the inhibition of type II phosphatidylinositol 4-kinase (PI4KII) activity and the enhancement of glutamate toxicity was blocked by a short fragment of Abeta, Abeta(31-35). Such protective effects of shorter fragments derived from Abeta(31-35) were examined in this study to reach the shortest effective peptide, using recombinant human PI4KII and primary cultured rat hippocampal neurons. Among the peptides tested (Abeta(31-34), Abeta(31-33), Abeta(31-32), Abeta(32-35), Abeta(33-35), Abeta(34-35), Abeta(32-34), Abeta(33-34) and Abeta(32-33)), Abeta(31-34), Abeta(32-35) and Abeta(32-34) blocked both the Abeta(1-42)-induced inhibition of PI4KII activity and enhancement of glutamate toxicity on cell viability. The shortest peptide among them, Abeta(32-34), showed a dose-dependent protective effect with 50% effective concentration near 1nM, while Abeta(34-32), with a reverse amino acid sequence for Abeta(32-34), showed no protective effects. Thus, a tripeptide, Abeta(32-34) i.e. Ile-Gly-Leu, may be available as a lead compound for designing effective Abeta antagonists.