Perlecan Binds to the β-Amyloid Proteins (Aβ) of Alzheimer's Disease, Accelerates Aβ Fibril Formation, and Maintains Aβ Fibril Stability

Abstract: Perlecan is a specific heparan sulfate proteoglycan that accumulates in the fibrillar β-amyloid (Aβ) deposits of Alzheimer's disease. Perlecan purified from the Engelbreth-Holm-Swarm tumor was used to define perlecan's interactions with Aβ and its effects on Aβ fibril formation. Using a solid-phase binding immunoassay, freshly solubilized full-length Aβ peptides bound immobilized perlecan at two sites, representing both high-affinity [K_D = ～5.8 × 10^?11M for Aβ (1–40); K_D = ～6.5 × 10^?12M for Aβ (1–42)] and lower-affinity [K_D = 3.5 × 10^?8M for Aβ (1–40); K_D = 4.3 × 10^?8M for Aβ (1–42)] interactions. An increase in the binding capacity of Aβ (1–40) to perlecan correlated with an increase in Aβ amyloid fibril formation during a 1-week incubation period. The high-capacity binding of Aβ (1–40) to perlecan was similarly observed using perlecan heparan sulfate glycosaminoglycans and was completely abolished by heparin, but not by chondroitin-4-sulfate. Using a thioflavin T fluorometry assay, perlecan accelerated the rate of Aβ (1–40) amyloid fibril formation, causing a significant increase in Aβ fibril assembly over a 2-week incubation period at 1 h (2.8-fold increase), 1 day (3.6-fold increase), and 3 days (2.8-fold increase) in comparison with Aβ (1–40) alone. Perlecan also initially accelerated the formation of Aβ (1–42) fibrils within 1 h and maintained significantly higher levels of Aβ (1–42) thioflavin T fluorescence throughout a 2-week experimental period in comparison with Aβ (1–42) alone, suggesting perlecan's ability to maintain amyloid fibril stability. Perlecan's effects on Aβ (1–40) fibril formation and maintenance of Aβ (1–42) fibril stability occurred in a dose-dependent manner and was also mediated primarily by perlecan's glycosaminoglycan chains. Perlecan was the most effective enhancer and accelerator of Aβ fibril formation when compared directly with other amyloid plaque components, including apolipoprotein E, α₁-antichymotrypsin, P component, C1q, and C3. This study, therefore, demonstrates that perlecan not only binds to the predominant isoforms of Aβ, but also accelerates Aβ fibril formation and stabilizes amyloid fibrils once formed, confirming pivotal roles for perlecan in the pathogenesis of Aβ amyloidosis in Alzheimer's disease.     

Neurodegeneration in an Aβ-induced model of Alzheimer's disease: the role of Cdk5

Cdk5 dysregulation is a major event in the neurodegenerative process of Alzheimer's disease (AD). In vitro studies using differentiated neurons exposed to Aβ exhibit Cdk5-mediated tau hyperphosphorylation, cell cycle re-entry and neuronal loss. In this study we aimed to determine the role of Cdk5 in neuronal injury occurring in an AD mouse model obtained through the intracerebroventricular (icv) injection of the Aβ_1–40 synthetic peptide. In mice icv-injected with Aβ, Cdk5 activator p35 is cleaved by calpains, leading to p25 formation and Cdk5 overactivation. Subsequently, there was an increase in tau hyperphosphorylation, as well as decreased levels of synaptic markers. Cell cycle reactivation and a significant neuronal loss were also observed. These neurotoxic events in Aβ-injected mice were prevented by blocking calpain activation with MDL28170 , which was administered intraperitoneally (ip). As MDL prevents p35 cleavage and subsequent Cdk5 overactivation, it is likely that this kinase is involved in tau hyperphosphorylation, cell cycle re-entry, synaptic loss and neuronal death triggered by Aβ. Altogether, these data demonstrate that Cdk5 plays a pivotal role in tau phosphorylation, cell cycle induction, synaptotoxicity, and apoptotic death in postmitotic neurons exposed to Aβ peptides in vivo , acting as a link between diverse neurotoxic pathways of AD.     

Aggregation and Metal-Binding Properties of Mutant Forms of the Amyloid Aβ Peptide of Alzheimer's Disease

Abstract: The fibrillogenic properties of Alzheimer's Aβ peptides corresponding to residues 1–40 of the normal human sequence and to two mutant forms containing the replacement Ala²¹ to Gly or Glu²² to Gln were compared. At pH 7.4 and 37°C the Gln²² peptide was found to aggregate and precipitate from solution faster than the normal Aβ, whereas the Gly²¹ peptide aggregated much more slowly. Electron microscopy showed that the aggregates all had fibrillar structures. Circular dichroism spectra of these peptides revealed that aggregation of the normal and Gln²² sequences was associated with spectral changes consistent with a transformation from random coil to β sheet, whereas the spectrum of the Gly²¹ peptide remained almost unchanged during a period in which little or no aggregation occurred. When immobilised by spotting onto nitrocellulose membranes the peptides bound similar amounts of the radioisotope ⁶⁵Zn²⁺. Of several competing metal ions, tested at 20× the concentration of Zn²⁺, Cu²⁺ displaced >95% of the radioactivity from all three peptides and Ni²⁺ produced >50% displacement in each case. Some other metal ions tested caused lesser displacement, but Fe²⁺ and Al³⁺ were without effect. In a saturation binding assay, a value of 3.2 µM was obtained for the binding of Zn²⁺ to Aβ but our data provided no evidence for a reported higher affinity site (107 nM). The results suggest that the neuropathology associated with the Gly²¹ mutation is not due to enhanced fibrillogenic or different metal-binding properties of the peptide and that the binding of zinc to amyloid peptides is not a specific phenomenon.     

Modulation of Aβ peptides by estrogen in mouse models

Clinical studies have shown that estrogen deprivation through menopause is a risk factor in both the initiation and progression of Alzheimer's disease (AD) and that estrogen replacement therapy may be protective. One of the major pathological features in the human AD brain is the senile plaque, a proteinaceous structure composed mainly of heterogeneous peptides collectively known as A-beta (A(beta)). In vitro studies have linked estrogen with A(beta) modulation, suggesting that one-way that estrogen depletion at menopause may exacerbate the features of AD is through A(beta) accumulation. To test this, two studies were performed on transgenic models of amyloidosis. Firstly, transgenic mice without detectable amyloid aggregates were subjected to ovariectomy and estradiol supplementation, and A(beta) levels were assessed. Secondly, the effects of estrogen modulation were assessed in mice at an age when plaques would be forming initially. Overall, A(beta) levels were higher in estrogen-deprived mice than intact mice, and this effect could be reversed through the administration of estradiol. These data suggest that, in vivo, estrogen depletion leads to the accumulation of A(beta) in the CNS, which can be reversed through replacement of estradiol. These results provide evidence that post-menopausal estrogen depletion may be linked to an increased risk of AD through A(beta) modulation.     

N-cadherin-based adhesion enhances Aβ release and decreases Aβ42/40 ratio

In neurons, Presenilin 1(PS1)/γ-secretase is located at the synapses, bound to N-cadherin. We have previously reported that N-cadherin-mediated cell–cell contact promotes cell-surface expression of PS1/γ-secretase. We postulated that N-cadherin-mediated trafficking of PS1 might impact synaptic PS1-amyloid precursor protein interactions and Aβ generation. In the present report, we evaluate the effect of N-cadherin-based contacts on Aβ production. We demonstrate that stable expression of N-cadherin in Chinese hamster ovary cells, expressing the Swedish mutant of human amyloid precursor protein leads to enhanced secretion of Aβ in the medium. Moreover, N-cadherin expression decreased Aβ_42/40 ratio. The effect of N-cadherin expression on Aβ production was accompanied by the enhanced accessibility of PS1/γ-secretase to amyloid precursor protein as well as a conformational change of PS1, as demonstrated by the fluorescence lifetime imaging technique. These results indicate that N-cadherin-mediated synaptic adhesion may modulate Aβ secretion as well as the Aβ_42/40 ratio via PS1/N-cadherin interactions.     

Laminin 1 Attenuates β-Amyloid Peptide Aβ(1-40) Neurotoxicity of Cultured Fetal Rat Cortical Neurons

A growing amount of evidence indicates the involvement of extracellular matrix components, especially laminins, in the development of Alzheimer's disease, although their role remains unclear. In this study, we clearly demonstrate that laminin 1 inhibits beta-amyloid peptide (Abeta)-induced neuronal cell death by preventing the fibril formation and interaction of the Abeta peptide with cell membranes. The presence of laminin at a laminin/Abeta peptide molar ratio of 1:800 significantly inhibits the Abeta-induced apoptotic events, together with inhibition of amyloid fibril formation. The inhibitory effects of laminin 1 were time- and dose-dependent, whereas laminin 2 had less effect on Abeta neurotoxicity. A preincubation of laminin and Abeta was not required to observe the protective effect of laminin, suggesting a direct interaction between laminin 1 and Abeta. Moreover, laminin had no effect on the toxicity of the fibrillar Abeta peptide, suggesting an interaction of laminin with nonfibrillar species of the Abeta peptide, sequestering the peptide in a soluble form. These data extend our understanding of laminin-dependent binding of Abeta and highlight the possible modulation role of laminin regarding Abeta aggregation and neurotoxicity in vivo.     

Vaccination with soluble Aβ oligomers generates toxicity-neutralizing antibodies

In recent studies of transgenic models of Alzheimer's disease (AD), it has been reported that antibodies to aged beta amyloid peptide 1-42 (Abeta(1-42)) solutions (mixtures of Abeta monomers, oligomers and amyloid fibrils) cause conspicuous reduction of amyloid plaques and neurological improvement. In some cases, however, neurological improvement has been independent of obvious plaque reduction, and it has been suggested that immunization might neutralize soluble, non-fibrillar forms of Abeta. It is now known that Abeta toxicity resides not only in fibrils, but also in soluble protofibrils and oligomers. The current study has investigated the immune response to low doses of Abeta(1-42) oligomers and the characteristics of the antibodies they induce. Rabbits that were injected with Abeta(1-42) solutions containing only monomers and oligomers produced antibodies that preferentially bound to assembled forms of Abeta in immunoblots and in physiological solutions. The antibodies have proven useful for assays that can detect inhibitors of oligomer formation, for immunofluorescence localization of cell-attached oligomers to receptor-like puncta, and for immunoblots that show the presence of SDS-stable oligomers in Alzheimer's brain tissue. The antibodies, moreover, were found to neutralize the toxicity of soluble oligomers in cell culture. Results support the hypothesis that immunizations of transgenic mice derive therapeutic benefit from the immuno-neutralization of soluble Abeta-derived toxins. Analogous immuno-neutralization of oligomers in humans may be a key in AD vaccines.     

Proinflammatory cytokine interferon-γ increases induction of indoleamine 2,3-dioxygenase in monocytic cells primed with amyloid β peptide 1–42: implications for the pathogenesis of Alzheimer's disease

Indoleamine 2,3-dioxygenase (IDO) is the rate-limiting enzyme of the kynurenine pathway of tryptophan metabolism, ultimately leading to production of the excitotoxin quinolinic acid (QUIN) by monocytic cells. In the Tg2576 mouse model of Alzheimer's disease, systemic inflammation induced by lipopolysaccharide leads to an increase in IDO expression and QUIN production in microglia surrounding amyloid plaques. We examined whether the IDO over-expression in microglia could be mediated by brain proinflammatory cytokines induced during the peripheral inflammation using THP-1 cells and peripheral blood mononuclear cells (PBMC) as models for microglia. THP-1 cells pre-treated with 5–25 μM amyloid β peptide (Aβ) (1–42) but not with Aβ (1–40) or Aβ (25–35) became an activated state as indicated by their morphological changes and enhanced adhesiveness. IDO expression was only slightly increased in the reactive cells but strongly enhanced following treatment with proinflammatory cytokine interferon-γ (IFN-γ) but not with interleukin-1β, tumor necrosis factor-α, or interleukin-6 at 100 U/mL. The concomitant addition of Aβ (1–42) with IFN-γ was totally ineffective, indicating that Aβ pre-treatment is prerequisite for a high IDO expression. The priming effect of Aβ (1–42) for the IDO induction was also observed for PBMC. These findings suggest that IFN-γ induces IDO over-expression in the primed microglia surrounding amyloid plaques.     

Spontaneous aggregation and cytotoxicity of the beta-amyloid Abeta1-40: a kinetic model

The time dependency of the spontaneous aggregation of the fibrillogenic -Amyloid peptide, A^1–40, was measured by turbidity, circular dichroism, HPLC, and fluorescence polarization. The results by all methods were comparable and they were most consistent with a kinetic model where the peptide first slowly forms an activated monomeric derivative (AM), which is the only species able to initiate, by tetramerization, the formation of linear aggregates. The anti-A antibody 6E10, raised against residues 1–17, at concentrations of 200–300 nM delayed significantly the aggregation of 50 M amyloid peptide. The anti–A antibody 4G8, raised against residues 17–24, was much less active in that respect, while the antibody A162, raised against the C-terminal residues 39–43 of the full-length A was totally inactive at those concentrations. Concomitant with the aggregation experiments, we also measured the time dependency of the A^1–40–induced toxicity toward SH-EP1 cells and hippocampal neurons, evaluated by SYTOX Green fluorescence, lactate dehydrogenase release, and activation of caspases. The extent of cell damage measured by all methods reached a maximum at the same time and this maximum coincided with that of the concentration of AM. According to the kinetic scheme, the latter is the only transient peptide species whose concentration passes through a maximum. Thus, it appears that the toxic species of A^1–40 is most likely the same transient activated monomer that is responsible for the nucleation of fibril formation. These conclusions should provide a structural basis for understanding the toxicity of A^1–40 in vitro and possibly in vivo.     

Selective modulation of amyloid-β peptide degradation by flurbiprofen, fenofibrate, and related compounds regulates Aβ levels

Gamma‐secretase modulators (GSMs) include selected non‐steroidal anti‐inflammatory drugs such as flurbiprofen that selectively lowers the neurotoxic amyloid‐β peptide Aβ_1–42. GSMs are attractive targets for Alzheimer’s disease, in contrast to ‘inverse GSMs,’ such as fenofibrate, which selectively increase the level of Aβ_1–42. A methodology for screening of Aβ modulating drugs was developed utilizing an Aβ‐producing neuroblastoma cell line stably transfected with mutant human amyloid precursor protein, immunoprecipitation of Aβ peptides, and mass spectroscopic quantitation of Aβ_1–37/Aβ_1–38/Aβ_1–40/Aβ_1–42 using an Aβ internal standard. The unexpected conclusion of this work was that in this system, drug effects are independent of γ‐secretase. The methodology recapitulated reported results for modulation of Aβ by GSMs. However, control experiments in which exogenous Aβ_1–40/Aβ_1–42 was added (i) to drug‐treated wild‐type cells or (ii) to conditioned media from these wild‐type cells, gave comparable patterns of Aβ modulation. These results, suggesting that drugs modulate the ability of cell‐derived factors to degrade Aβ, was interrogated by adding protease inhibitors and performing molecular weight cut‐off fractionation. The results confirmed that modulation of Aβ_1–40/Aβ_1–42 was mediated by selective proteolysis. Treatment of N2a cells with flurbiprofen or fenofibric acid selectively enhanced Aβ_1–42 clearance by extracellular proteolysis; treatment with HCT‐1026 or fenofibrate (esters of flurbiprofen and fenobric acid) inhibited clearance of Aβ_1–40 and Aβ_1–42.     

Iron (III) induces aggregation of hyperphosphorylated tau and its reduction to iron (II) reverses the aggregation: implications in the formation of neurofibrillary tangles of Alzheimer's disease

Iron as well as aluminum is reported to accumulate in neurons with neurofibrillary tangles (NFTs) of Alzheimer's disease (AD) brain. Previously we demonstrated that aluminum (III) shows phosphate-dependent binding with hyperphosphorylated tau (PHFtau), the major constituent of NFTs, thereby inducing aggregation of PHFtau. Herein we report that iron (III) can also induce aggregation of soluble PHFtau. Importantly, for the aggregation of PHFtau to occur, iron in the oxidized state (III) is essential since iron in the reduced state (II) lacks such ability. Furthermore, iron (III)-induced aggregation is reversed by reducing iron (III) to iron (II). Thus the iron-participating aggregation is mediated not only by tau phosphorylation but also by the transition of iron between reduced (II) and oxidized (III) states. Further incubation of insoluble PHFtau aggregates isolated from AD brain with reducing agents produced liberation of solubilized PHFtau and iron (II), indicating that PHFtau in association with iron (III) constitutes the insoluble pool of PHFtau. These results indicate that iron might play a role in the aggregation of PHFtau leading to the formation of NFTs in AD brain.     

Alzheimer Aβ Amyloid Forms an Inhibitory Neuronal Substrate

Abstract: Alzheimer's disease (AD) is identified by the accumulation of amyloid plaques, neurofibrillary degeneration, and the accompanying neuronal loss. AD amyloid assembles into compact fibrous deposits from the amyloid β(Aβ) protein, which is a proteo-lytic fragment of the membrane-associated amyloid precursor protein. To examine the effects of amyloid on neuron growth, a hybrid mouse motoneuron cell line (NSC34) exhibiting spontaneous process formation was exposed to artificial "plaques" created from aggregated synthetic Aβ peptides. These correspond to full-length Aβ residues 1–40 (Aβ1–40), an internal β-sheet region comprising residues 11–28 (Aβ11–28), and a proposed toxic fragment comprising residues 25–35 (Aβ25–35). Fibers were immobilized onto culture dishes, and addition of cells to these in vitro plaques revealed that Aβ was not a permissive substrate for cell adhesion. Neurites in close contact with these deposits displayed abnormal swelling and a tendency to avoid contact with the Aβ fibers. In contrast, Aβ did not affect the adhesion or growth of rat astrocytes, implicating a specific Aβ-neuron relationship. The inhibitory effects were also unique to Aβ as no response was observed to deposits of pancreatic islet amyloid poly-peptide fibers. Considering the importance of cell adhesion in neurite elongation and axonal guidance, the antiadhesive properties of Aβ amyloid plaques found in vivo may contribute to the neuronal loss responsible for the clinical manifestations of AD.     

Aβ43 is more frequent than Aβ40 in amyloid plaque cores from Alzheimer disease brains

One hallmark of Alzheimer disease (AD) is the extracellular deposition of the amyloid β-peptide (Aβ) in senile plaques. Two major forms of Aβ are produced, 40 (Aβ40) and 42 (Aβ42) residues long. The most abundant form of Aβ is Aβ40, while Aβ42 is more hydrophobic and more prone to form toxic oligomers and the species of particular importance in early plaque formation. Thus, the length of the hydrophobic C-terminal seems to be very important for the oligomerization and neurotoxicity of the Aβ peptide. Here we investigated which Aβ species are deposited in AD brain. We analyzed plaque cores, prepared from occipital and frontal cortex, from sporadic and familial AD cases and performed a quantitative study using Aβ standard peptides. Cyanogen bromide was used to generate C-terminal Aβ fragments, which were analyzed by HPLC coupled to an electrospray ionisation ion trap mass spectrometer. We found a longer peptide, Aβ43, to be more frequent than Aβ40. No variants longer than Aβ43 could be observed in any of the brains. Immunohistochemistry was performed and was found to be in line with our findings. Aβ1-43 polymerizes rapidly and we suggest that this variant may be of importance for AD.     

Intracellular Accumulation of Detergent-Soluble Amyloidogenic Aβ Fragment of Alzheimer's Disease Precursor Protein in the Hippocampus of Aged Transgenic Mice

To study amyloid beta-protein (A beta) production and aggregation in vivo, we created two transgenic (Tg) mouse lines expressing the C-terminal 100 amino acids of human amyloid precursor protein (APP): Tg C100.V717F and Tg C100.WT. Western blot analysis showed that human APP-C100 and A beta were produced in brain and some peripheral tissues and A beta was produced in serum. Using antibodies specific for the A beta C terminus we found that Tg C100.V717F produced a 1.6-fold increase in A beta42/A beta40 compared with Tg C100.WT. Approximately 30% of total brain A beta (approximately 122 ng/g of wet tissue) was water-soluble. The remaining 70% of A beta partitioned into the particulate fraction and was completely sodium dodecyl sulfate-soluble. In contrast, human Alzheimer's disease brain has predominantly sodium dodecyl sulfate-insoluble A beta. Immunohistochemistry with an A beta(5-8) antibody showed that A beta or A beta-containing fragments accumulated intracellularly in the hippocampus of aged Tg C100.V717F mice. The soluble A beta levels in Tg brain are similar to those in normal human brain, and this may explain the lack of microscopic amyloid deposits in the Tg mice. However, this mouse model provides a system to study the intracellular processing and accumulation of A beta or A beta-containing fragments and to screen for compounds directed at the gamma-secretase activity.     

Beta-amyloid production,aggregation, and clearance as targets for therapy in Alzheimer's disease

1. Despite major efforts aimed at elucidating the molecular basis and physiopathology of Alzheimer's disease (AD), there is still no effective treatment available for this devastating disorder. The biological mechanisms underlying the development of AD are complex, as multiple factors appear to modulate (either positively or negatively) the progression of neurodegeneration in the brains of AD patients. Not surprisingly, a number of different therapeutic approaches aimed at distinct aspects of the disease are currently being pursued. Given its central role in the neuropathology of AD, the -amyloid peptide (A) is the focus of many such approaches.2. In this review, we discuss recent developments along three major lines of investigation: (i) identification and characterization of inhibitors of the enzymes involved in proteolytic processing of the amyloid precursor protein and production of A (ii) identification of the pathways involved in cerebral degradation and clearance of A and (iii) characterization of small-molecule inhibitors of amyloid aggregation that prevent cerebral amyloid deposition and neurotoxicity.3. Significant progress has been achieved in these directions, opening up new perspectives toward the development of effective approaches for the treatment or prevention of AD.     

Mutations that reduce aggregation of the Alzheimer's Abeta42 peptide: an unbiased search for the sequence determinants of Abeta amyloidogenesis

The primary component of amyloid plaque in the brains of Alzheimer's patients is the 42 residue amyloid-beta-peptide (Abeta42). Although the amino acid residue sequence of Abeta42 is known, the molecular determinants of Abeta amyloidogenesis have not been elucidated. To facilitate an unbiased search for the sequence determinants of Abeta aggregation, we developed a genetic screen that couples a readily observable phenotype in E. coli to the ability of a mutation in Abeta42 to reduce aggregation. The screen is based on our finding that fusions of the wild-type Abeta42 sequence to green fluorescent protein (GFP) form insoluble aggregates in which GFP is inactive. Cells expressing such fusions do not fluoresce. To isolate variants of Abeta42 with reduced tendencies to aggregate, we constructed and screened libraries of Abeta42-GFP fusions in which the sequence of Abeta42 was mutated randomly. Cells expressing GFP fusions to soluble (non-aggregating) variants of Abeta42 exhibit green fluorescence. Implementation of this screen enabled the isolation of 36 variants of Abeta42 with reduced tendencies to aggregate. The sequences of most of these variants are consistent with previous models implicating hydrophobic regions as determinants of Abeta42 aggregation. Some of the variants, however, contain amino acid substitutions not implicated in pre-existing models of Abeta amyloidogenesis.     

CD40 signaling and Alzheimer's disease pathogenesis

The interaction between CD40 and its cognate ligand, CD40 ligand, is a primary regulator of the peripheral immune response, including modulation of T lymphocyte activation, B lymphocyte differentiation and antibody secretion, and innate immune cell activation, maturation, and survival. Recently, we and others have identified CD40 expression on a variety of CNS cells, including endothelial cells, smooth muscle cells, astroglia and microglia, and have found that, on many of these cells, CD40 expression is enhanced by pro-inflammatory stimuli. Importantly, the CD40–CD40 ligand interaction on microglia triggers a series of intracellular signaling events that are discussed, beginning with Src-family kinase activation and culminating in microglial activation as evidenced by tumor necrosis factor- secretion. Based on the involvement of microglial activation and brain inflammation in Alzheimer's disease pathogenesis, we have investigated co-stimulation of microglia, smooth muscle, and endothelial cells with CD40 ligand in the presence of low doses of freshly solubilized amyloid-β peptides. Data reviewed herein show that CD40 ligand and amyloid-β act synergistically to promote pro-inflammatory responses by these cells, including secretion of interleukin-1β by endothelial cells and tumor necrosis factor- by microglia. As these cytokines have been implicated in neuronal injury, a comprehensive model of pro-inflammatory CD40 ligand and amyloid-β initiated Alzheimer's disease pathogenesis (mediated by multiple CNS cells) is proposed.     

Aggregation of β-Amyloid Peptide Is Promoted by Membrane Phospholipid Metabolites Elevated in Alzheimer's Disease Brain

Abstract: Increased amounts of β-amyloid (Aβ) peptide deposits are found in Alzheimer's disease brain. These amyloid deposits have been implicated in the pathophysiology of this common dementing illness. Aβ peptides have been shown to be toxic to neurons in cell culture, and this toxicity is critically dependent on the aggregation of the peptide into cross-β-pleated sheet fibrils. Also, in vivo and postmortem NMR studies have shown changes in certain brain membrane phospholipid metabolites in normal aging and more extensive alterations in patients with Alzheimer's disease. The finding that membrane phospholipids affect the aggregation of Aβ suggests that the abnormalities in membrane metabolism found in Alzheimer's disease could affect the deposition of Aβ in vivo. Therefore, we examined the effect of membrane phospholipid metabolites that are altered in Alzheimer's disease brain on the aggregation of Aβ(1–40) using a light scattering method. Certain metabolites (glycerophosphocholine, glycerophosphoethanolamine, and α-glycerophosphate) augment the aggregation of Aβ. Other membrane phospholipid metabolites (phosphocholine, phosphoethanolamine, and inositol-1-phosphate) have no effect. We conclude that increased membrane phospholipid metabolite concentrations may play a role in the deposition of Aβ seen in normal aging and the even greater deposition of Aβ observed in Alzheimer's disease.     

The glial glutamate transporter, GLT-1, is oxidatively modified by 4-hydroxy-2-nonenal in the Alzheimer's disease brain: the role of Aβ1–42

Glutamate transporters are involved in the maintenance of synaptic glutamate concentrations. Because of its potential neurotoxicity, clearance of glutamate from the synaptic cleft may be critical for neuronal survival. Inhibition of glutamate uptake from the synapse has been implicated in several neurodegenerative disorders. In particular, glutamate uptake is inhibited in Alzheimer's disease (AD); however, the mechanism of decreased transporter activity is unknown. Oxidative damage in brain is implicated in models of neurodegeneration, as well as in AD. Glutamate transporters are inhibited by oxidative damage from reactive oxygen species and lipid peroxidation products such as 4-hydroxy-2-nonenal (HNE). Therefore, we have investigated a possible connection between the oxidative damage and the decreased glutamate uptake known to occur in AD brain. Western blots of immunoprecipitated HNE-immunoreactive proteins from the inferior parietal lobule of AD and control brains suggest that HNE is conjugated to GLT-1 to a greater extent in the AD brain. A similar analysis of beta amyloid (Abeta)-treated synaptosomes shows for the first time that Abeta1-42 also increases HNE conjugation to the glutamate transporter. Together, our data provide a possible link between the oxidative damage and neurodegeneration in AD, and supports the role of excitotoxicity in the pathogenesis of this disorder. Furthermore, our data suggests that Abeta may be a possible causative agent in this cascade.