Alzheimer's Disease

Peptide aldehyde inhibitors of the chymotrypsin-like activity of the proteasome (CLIP) such as N-acetyl-Leu-Leu-Nle-H (or ALLN) have been shown previously to inhibit the secretion of beta-amyloid peptide (A beta) from cells. To evaluate more fully the role of the proteasome in this process, we have tested the effects on A beta formation of a much wider range of peptide-based inhibitors of CLIP than published previously. The inhibitors tested included several peptide boronates, some of which proved to be the most potent peptide-based inhibitors of beta-amyloid production reported so far. We found that the ability of the peptide aldehyde and boronate inhibitors to suppress A beta formation from cells correlated extremely well with their potency as CLIP inhibitors. Thus, we conclude that the proteasome may be involved either directly or indirectly in A beta formation.     

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.     

Differences Between Vascular and Plaque Core Amyloid in Alzheimer's Disease

Abstract: The predominant protein of cerebrovascular and plaque core amyloid in Alzheimer's disease, Down's syndrome, hereditary hemorrhage with amyloidosis—Dutch type, sporadic cerebral amyloid angiopathy, and age-related amyloidosis is a unique polypeptide, called β protein. The length of the plaque amyloid protein was reported to be 42–43 residues, but the complete length of the cerebral vascular amyloid is not known. To clarify this issue, amyloid fibrils from the leptomeninges of an Alzheimer's disease patient were isolated and the primary structure determined. The complete sequence of cerebrovascular β-amyloid protein, although homologous to the plaque core amyloid protein previously reported, has 39 residues instead of 42. Amino terminal heterogeneity is present but minimal, and it is three residues shorter at the carboxy terminus. These differences are similar to those found in two cases of hereditary hemorrhage with amyloidosis—Dutch type. The differences between vascular and plaque β-amyloid may reflect diverse processing of the β protein precursor in the vessel wall and brain parenchyma due to tissue-specific endopeptidases.     

α-Secretase-Derived Product of β-Amyloid Precursor Protein Is Decreased by Presenilin 1 Mutations Linked to Familial Alzheimer's Disease

Abstract: Recent reports indicate that missense mutations on presenilin (PS) 1 are likely responsible for the main early-onset familial forms of Alzheimer's disease (FAD). Consensual data obtained through distinct histopathological, cell biology, and molecular biology approaches have led to the conclusion that these PS1 mutations clearly trigger an increased production of the 42-amino-acid-long species of β-amyloid peptide (Aβ). Here we show that overexpression of wild-type PS1 in HK293 cells increases Aβ40 secretion. By contrast, FAD-linked mutants of PS1 trigger increased secretion of both Aβ40 and Aβ42 but clearly favor the production of the latter species. We also demonstrate that overexpression of the wild-type PS1 augments the α-secretase-derived C-terminally truncated fragment of β-amyloid precursor protein (APPα) recovery, whereas transfectants expressing mutated PS1 secrete drastically lower amounts of APPα when compared with cells expressing wild-type PS1. This decrease was also observed when comparing double transfectants overexpressing wild-type β-amyloid precursor protein and either PS1 or its mutated congener M146V-PS1. Altogether, our data indicate that PS mutations linked to FAD not only trigger an increased ratio of Aβ42 over total Aβ secretion but concomitantly down-regulate the production of APPα.     

α2-Macroglobulin as a β-Amyloid Peptide-Binding Plasma Protein

Abstract: The β-amyloid peptide (Aβ) is a normal proteolytic processing product of the amyloid precursor protein, which is constitutively expressed by many, if not most, cells. For reasons that are still unclear, Aβ is deposited in an aggregated fibrillar form in both diffuse and senile plaques in the brains of patients with Alzheimer's disease (AD). The factor(s) responsible for the clearance of soluble Aβ from biological fluids or tissues are poorly understood. We now report that human α₂-macroglobulin (α₂M), a major circulating endoproteinase inhibitor, which has recently been shown to be present in senile plaques in AD, binds ¹²⁵I-Aβ_(1–42) with high affinity (apparent dissociation constant of 3.8 × 10^?10M). Approximately 1 mol of Aβ is bound per mole of α₂M. Both native and methylamine-activated α₂M bind ¹²⁵I-Aβ_(1–42). The binding of ¹²⁵I-Aβ_(1–42) to α₂M is enhanced by micromolar concentrations of Zn²⁺ (but not Ca²⁺) and is inhibited by noniodinated Aβ_(1–42) and Aβ_(1–40) but not by the reverse peptide Aβ_(40-1) or the cytokines interleukin 1β or interleukin 2. α₁-Antichymotrypsin, another plaque-associated protein, inhibits both the binding of ¹²⁵I-Aβ_(1–42) to α₂M as well as the degradation of ¹²⁵I-Aβ_(1–42) by proteinase-activated α₂M. Moreover, the binding of ¹²⁵I-Aβ_(1–42) to α₂M protects the peptide from proteolysis by exogenous trypsin. These data suggest that α₂M may function as a carrier protein for Aβ and could serve to either facilitate or impede clearance of Aβ from tissues such as the brain.     

β-Amyloid Peptide Interacts Specifically with the Carboxyl-Terminal Domain of Human Apolipoprotein E

Abstract : Growing evidence indicates the involvement of apolipoprotein E (apoE) in the development of late-onset and sporadic forms of Alzheimer's disease, although its exact role remains unclear. We previously demonstrated that β-amyloid peptide (Aβ) displays membrane-destabilizing properties and that only apoE2 and E3 isoforms inhibit these properties. In this study, we clearly demonstrate that the carboxy-terminal lipid-binding domain of apoE (e.g., residues 200-299) is responsible for the Aβ-binding activity of apoE and that this interaction involves pairs of apoE amphipathic α-helices. We further demonstrate that Aβ is able to inhibit the association of the C-terminal domain of apoE with lipids due to the formation of Aβ/apoE complexes resistant to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. On the contrary, the amino-terminal receptor-binding domain of apoE (e.g., residues 129-169) is not able to form stable complexes with Aβ. These data extend our understanding of human apoE-dependent binding of Aβ by involving the C-terminal domain of apoE in the efficient formation of apoE/Aβ complex.     

β-Amyloid1–40 Increases Expression of β-Amyloid Precursor Protein in Neuronal Hybrid Cells

Abstract: Studies of cell injury and death in Alzheimer's disease have suggested a prominent role for β-amyloid peptide (β-AP), a 40–43-amino-acid peptide derived from a larger membrane glycoprotein, β-amyloid precursor protein (β-APP). Previous experiments have demonstrated that β-AP induces cytotoxicity in a neuronal hybrid cell line (MES 23.5) in vitro. Here, we demonstrate that β-APP mRNA content is increased 3.5-fold in 24 h after treatment with β-AP_1–40. Accompanying β-AP_1–40-induced cell injury, levels of cell-associated β-APP and a C-terminal intermediate fragment are increased up to 15-fold, and levels of secreted forms of β-APP and 12- and 4-kDa fragments are also increased. Application of β-APP antisense oligodeoxynucleotide reduces both cytotoxicity and β-APP expression. 6-Hydroxydopamine application or glucose deprivation causes extensive cell damage, but they do not increase β-APP expression. These results suggest a selective positive feedback mechanism whereby β-AP may induce cytotoxicity and increase levels of potentially neurotrophic as well as amyloidogenic fragments of β-APP with the net consequence of further neuronal damage.     

β-Amyloid-Induced Neurotoxicity of a Hybrid Septal Cell Line Associated with Increased Tau Phosphorylation and Expression of β-Amyloid Precursor Protein

Abstract: Recent evidence suggests that β-amyloid peptide (β-AP) may induce tau protein phosphorylation, resulting in loss of microtubule binding capacity and formation of paired helical filaments. The mechanism by which β-AP increases tau phosphorylation, however, is unclear. Using a hybrid septal cell line, SN56, we demonstrate that aggregated β-AP_1–40 treatment caused cell injury. Accompanying the cell injury, the levels of phosphorylated tau as well as total tau were enhanced as detected immunochemically by AT8, PHF-1, Tau-1, and Tau-5 antibodies. Alkaline phosphatase treatment abolished AT8 and PHF-1 immunoreactivity, confirming that the tau phosphorylation sites were at least at Ser^199/202 and Ser³⁹⁶. In association with the increase in tau phosphorylation, the immunoreactivity of cell-associated and secreted β-amyloid precursor protein (β-APP) was markedly elevated. Application of antisense oligonucleotide to β-APP reduced expression of β-APP and immunoreactivity of phosphorylated tau. Control peptide β-AP_1–28 did not produce significant effects on tau phosphorylation, although it slightly increased cell-associated β-APP. These results suggest that βAP_1–40-induced tau phosphorylation may be associated with increased β-APP expression in degenerated neurons.     

Isoform-Specific Effects of Apolipoproteins E2, E3, and E4 on Cerebral Capillary Sequestration and Blood-Brain Barrier Transport of Circulating Alzheimer's Amyloid β

Abstract: Cerebral capillary sequestration and blood-brain barrier (BBB) permeability to apolipoproteins E2 (apoE2), E3 (apoE3), and E4 (apoE4) and to their complexes with sAβ_1–40, a peptide homologous to the major form of soluble Alzheimer's amyloid β, were studied in perfused guinea pig brain. Cerebrovascular uptake of three apoE isoforms was low, their blood-to-brain transport undetectable, but uptake by the choroid plexus significant. Binding of all three isoforms to sAβ_1–40 in vitro was similar with a K _D between 11.8 and 12.9 n M . Transport into brain parenchyma and sequestration by BBB and choroid plexus were negligible for sAβ_1–40-apoE2 and sAβ_1–40-apoE3, but significant for sAβ_1–40-apoE4. After 10 min, 85% of sAβ_1–40-apoE4 taken up at the BBB remained as intact complex, whereas free sAβ_1–40 was 51% degraded. Circulating apoE isoforms have contrasting effects on cerebral capillary uptake of and BBB permeability of sAβ. ApoE2 and apoE3 completely prevent cerebral capillary sequestration and blood-to-brain transport of sAβ_1–40. Conversely, apoE4, by entering brain microvessels and parenchyma as a stable complex with sAβ, reduces peptide degradation and may predispose to cerebrovascular and possibly enhance parenchymal amyloid formation under pathological conditions.     

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.     

β-Amyloid Precursor Protein Isoforms in Various Rat Brain Regions and During Brain Development

To address the question of the possible functions of different Alzheimer's disease beta-amyloid precursor protein (beta-APP) isoforms in the brain, we studied their expression at different times during postnatal rat brain development and in various regions of the adult rat brain. Polyclonal antibodies directed to two peptide antigens were used. The majority of all beta-APP forms was found to be soluble as revealed by western blot analysis. The highest level of most beta-APP forms was reached in the second postnatal week, which is the time of brain maturation and completion of synaptic connections. Strikingly high concentrations of the Kunitz protease inhibitor-containing beta-APP were present in the adult olfactory bulb, where continuous synaptogenesis occurs in the adult animal. These findings support the idea of an involvement of beta-APPs in the processes of cell differentiation and, probably, in the establishment of synaptic contacts.     

Secreted Forms of β-Amyloid Precursor Protein Protect Against Ischemic Brain Injury

Abstract: The β-amyloid precursor protein (βAPP) is the source of the amyloid β-peptide that accumulates in the brain in Alzheimer's disease. A major processing pathway for βAPP involves an enzymatic cleavage within the amyloid β-peptide sequence that liberates secreted forms of βAPP (APP^Ss) into the extracellular milieu. We now report that postischemic administration of these APP^Ss intracerebroventricularly protects neurons in the CA1 region of rat hippocampus against ischemic injury. Treatment with APP^S695 or APP^S751 resulted in increased neuronal survival, and the surviving cells were functional as demonstrated by their ability to synthesize protein. These data provide direct evidence for a neuroprotective action of APP^Ss in vivo.     

Characterization of the Binding of Amyloid-β Peptide to Cell Culture-Derived Native Apolipoprotein E2, E3, and E4 Isoforms and to Isoforms from Human Plasma

Abstract: The ε4 allele of apolipoprotein E (apoE, protein; APOE, gene) is a major risk factor for Alzheimer's disease (AD). Genetically, the frequency of the ε4 allele is enriched in early-onset sporadic, late-onset familial, and common late-onset sporadic AD. ApoE is found in the extracellular amyloid-β (Aβ) deposits that are characteristic features of AD. In this study, we examined the interaction between Aβ and apoE isoforms. The apoE isoforms used in this study were either produced by stably transfected Chinese hamster ovary cells (CHO) or were from human plasma. We report that when similar concentrations of the apoE isoforms were used, native nonpurified apoE3 from recombinant CHO-derived sources bound Aβ, but apoE4 did not. In fact, in our system, binding of recombinant apoE4 to Aβ was never detectable, even after incubation for 4 days. Furthermore, using the same assay conditions, native apoE2, like apoE3, binds Aβ avidly. Furthermore, when human plasma apoE isoforms are tested in Aβ binding experiments, apoE3 bound Aβ more avidly than apoE4, and a major apoE/Aβ complex (the 40-kDa form) was observed with plasma apoE3 but not apoE4. These data extend our understanding of apoE isoform-dependent binding of Aβ by associating apoE2 with efficient apoE/Aβ complex formation and demonstrate that native apoE3 (whether recombinant or derived from human plasma) forms sodium dodecyl sulfate-stable apoE/Aβ complexes more readily than native apoE4. The different Aβ-binding properties of native apoE4 versus native apoE3 provide insight into the molecular mechanisms by which the APOE ε4 allele exerts its risk factor effects in AD.     

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.     

Surface Phosphorylation by Ecto-Protein Kinase C in Brain Neurons: A Target for Alzheimer's β-Amyloid Peptides

Abstract: The powerful regulatory machinery of protein phosphorylation operates in the extracellular environment of the brain. Enzymatic activity with the catalytic specificity of protein kinase C (PKC) was detected on the surface of brain neurons, where it can serve as a direct target for neurotrophic and neurotoxic substances that control neuronal development and cause neurodegeneration. This activity fulfilled all the criteria required of an ectoprotein kinase (ecto-PK). Detailed analysis of surface protein phosphorylation in cultured brain neurons using specific exogenous substrates (casein, histones, and myelin basic protein), inhibitors (PKC-pseudosubstrate 19–36; K252b) and antibodies (anti-PKC catalytic region M.Ab.1.9, antibodies to the carboxy-terminus of eight PKC isozymes) revealed several types of ecto-PK activity, among them ecto-PKs with catalytic specificity of the PKC isozymes ζ and δ. The activity of the neuronal ecto-PKC is constitutive and not stimulated by phorbol esters. The phosphorylation of a 12K/13K surface protein duplex by ecto-PKC-δ was found to be developmentally regulated, with peak activity occurring during the onset of neuritogenesis. Alzheimer's amyloid peptides β1–40 and β25–35 applied at neurotrophic concentrations stimulated the phosphorylation of endogenous substrates of ecto-PKC activity in brain neurons but inhibited specifically this surface phosphorylation activity with the same dose-response relationships that cause neurodegeneration. As may be expected from a relevant pathophysiological activity, β-amyloid peptide 1–28 did not inhibit this surface phosphorylation. The discovery that ecto-PKC-mediated protein phosphorylation serves as a target for β-amyloid peptides at the very site they operate, i.e., at the neuronal cell surface, opens a new research direction in the investigation of molecular events that play a role in the etiology of developmental disabilities and neurodegenerative disorders.     

Molecular Determinants of the Interaction Between the C-Terminal Domain of Alzheimer's β-Amyloid Peptide and Apolipoprotein E α-Helices

In a previous work, we predicted and demonstrated that the 29-42-residue fragment of beta-amyloid peptide (Abeta peptide) has in vitro capacities close to those of the tilted fragment of viral fusion proteins. We further demonstrated that apolipoprotein E2 and E3 but not apolipoprotein E4 can decrease the fusogenic activity of Abeta(29-42) via a direct interaction. Therefore, we suggested that this fragment is implicated in the neurotoxicity of Abeta and in the protective effects of apolipoprotein E in Alzheimer's disease. Because structurally related apolipoproteins do not interact with the Abeta C-terminal domain but inhibit viral fusion, we suggested that interactions existing between fusogenic peptides and apolipoproteins are selective and responsible for the inhibition of fusion. In this study, we simulated interactions of all amphipathic helices of apolipoproteins E and A-I with Abeta and simian immunodeficiency virus (SIV) fusogenic fragments by molecular modeling. We further calculated cross-interactions that do not inhibit fusion in vitro. The results suggest that interactions of hydrophobic residues are the major event to inhibit the fusogenic capacities of Abeta(29-42) and SIV peptides. Selectivity of those interactions is due to the steric complementarity between bulky hydrophobic residues in the fusogenic fragments and hydrophobic residues in the apolipoprotein C-terminal amphipathic helices.     

Apoptotic Cell Death Induced by β-Amyloid1–42 Peptide Is Cell Type Dependent

Abstract: β-Amyloid peptide (Aβ), a proteolytic fragment of the β-amyloid precursor protein, is a major component of senile plaques in the brain of Alzheimer's disease patients. This neuropathological feature is accompanied by increased neuronal cell loss in the brain and there is evidence that Aβ is directly neurotoxic. In the present study reduced cell viability in four different neuroblastoma cell types was observed after treatment with human Aβ_1–42 for 1 day. Of the cell types tested rat PC12 and human IMR32 cells were most susceptible to Aβ toxicity. Chromosomal condensation and fragmentation of nuclei were seen in PC12, NB₂a, and B104 cells but not in IMR32 cells irrespective of their high sensitivity to Aβ. Electrophoretic analysis of cellular DNA confirmed internucleosomal DNA fragmentation typical for apoptosis in all cell types except IMR32. These findings suggest that the form of Aβ-induced cell death (necrosis or apoptosis) may depend on the cell type.     

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.