Protein phosphatase 5 protects neurons against amyloid-β toxicity

Amyloid-β (Aβ) is thought to promote neuronal cell loss in Alzheimer's disease, in part through the generation of reactive oxygen species (ROS) and subsequent activation of mitogen-activated protein kinase (MAPK) pathways. Protein phosphatase 5 (PP5) is a ubiquitously expressed serine/threonine phosphatase which has been implicated in several cell stress response pathways and shown to inactivate MAPK pathways through key dephosphorylation events. Therefore, we examined whether PP5 protects dissociated embryonic rat cortical neurons in vitro from cell death evoked by Aβ. As predicted, neurons in which PP5 expression was decreased by small-interfering RNA treatment were more susceptible to Aβ toxicity. In contrast, over-expression of PP5, but not the inactive mutant, PP5(H304Q), prevented MAPK phosphorylation and neurotoxicity induced by Aβ. PP5 also prevented cell death caused by direct treatment with H₂O₂, but did not prevent Aβ-induced production of ROS. Thus, the neuroprotective effect of PP5 requires its phosphatase activity and lies downstream of Aβ-induced generation of ROS. In summary, our data indicate that PP5 plays a pivotal neuroprotective role against cell death induced by Aβ and oxidative stress. Consequently, PP5 might be an effective therapeutic target in Alzheimer's disease and other neurodegenerative disorders in which oxidative stress is implicated.     

Zn(II)- and Cu(II)-induced non-fibrillar aggregates of amyloid-β (1–42) peptide are transformed to amyloid fibrils, both spontaneously and under the influence of metal chelators

Aggregation of amyloid-β (Aβ) peptides is a central phenomenon in Alzheimer's disease. Zn(II) and Cu(II) have profound effects on Aβ aggregation; however, their impact on amyloidogenesis is unclear. Here we show that Zn(II) and Cu(II) inhibit Aβ₄₂ fibrillization and initiate formation of non-fibrillar Aβ₄₂ aggregates, and that the inhibitory effect of Zn(II) (IC₅₀ = 1.8 μmol/L) is three times stronger than that of Cu(II). Medium and high-affinity metal chelators including metallothioneins prevented metal-induced Aβ₄₂ aggregation. Moreover, their addition to preformed aggregates initiated fast Aβ₄₂ fibrillization. Upon prolonged incubation the metal-induced aggregates also transformed spontaneously into fibrils, that appear to represent the most stable state of Aβ₄₂. H13A and H14A mutations in Aβ₄₂ reduced the inhibitory effect of metal ions, whereas an H6A mutation had no significant impact. We suggest that metal binding by H13 and H14 prevents the formation of a cross-β core structure within region 10–23 of the amyloid fibril. Cu(II)-Aβ₄₂ aggregates were neurotoxic to neurons in vitro only in the presence of ascorbate, whereas monomers and Zn(II)-Aβ₄₂ aggregates were non-toxic. Disturbed metal homeostasis in the vicinity of zinc-enriched neurons might pre-dispose formation of metal-induced Aβ aggregates, subsequent fibrillization of which can lead to amyloid formation. The molecular background underlying metal-chelating therapies for Alzheimer's disease is discussed in this light.     

Binding of zinc(II) and copper(II) to the full-length Alzheimer's amyloid-beta peptide

There is evidence that binding of metal ions like Zn2+ and Cu2+ to amyloid beta-peptides (Abeta) may contribute to the pathogenesis of Alzheimer's disease. Cu2+ and Zn2+ form complexes with Abeta peptides in vitro; however, the published metal-binding affinities of Abeta vary in an enormously large range. We studied the interactions of Cu2+ and Zn2+ with monomeric Abeta(40) under different conditions using intrinsic Abeta fluorescence and metal-selective fluorescent dyes. We showed that Cu(2+) forms a stable and soluble 1 : 1 complex with Abeta(40), however, buffer compounds act as competitive copper-binding ligands and affect the apparent K(D). Buffer-independent conditional K(D) for Cu(II)-Abeta(40) complex at pH 7.4 is equal to 0.035 micromol/L. Interaction of Abeta(40) with Zn2+ is more complicated as partial aggregation of the peptide occurs during zinc titration experiment and in the same time period (within 30 min) the initial Zn-Abeta(40) complex (K(D) = 60 micromol/L) undergoes a transition to a more tight complex with K(D) approximately 2 micromol/L. Competition of Abeta(40) with ion-selective fluorescent dyes Phen Green and Zincon showed that the K(D) values determined from intrinsic fluorescence of Abeta correspond to the binding of the first Cu2+ and Zn2+ ions to the peptide with the highest affinity. Interaction of both Zn2+ and Cu2+ ions with Abeta peptides may occur in brain areas affected by Alzheimer's disease and Zn2+-induced transition in the peptide structure might contribute to amyloid plaque formation.     

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.     

Aminopeptidase A contributes to the N-terminal truncation of amyloid β-peptide

Several lines of data previously indicated that N‐terminally truncated forms of amyloid‐β (Aβ) peptides are likely the earliest and more abundant species immunohistochemically detectable in Alzheimer’s disease‐affected brains. It is noteworthy that the free N‐terminal residue of full‐length Aβ (fl‐Aβ) is an aspartyl residue, suggesting that Aβ could be susceptible to exopeptidasic attack by aminopeptidase A (APA)‐like proteases. In this context, we have examined whether APA could target Aβ peptides in both cell‐free and cellular models. We first show that the general aminopeptidase inhibitor amastatin as well as two distinct aminopeptidase A inhibitors EC33 and pl302 both significantly increase the recovery of genuine fl‐Aβ peptides generated by cells over‐expressing Swedish‐mutated β amyloid precursor protein (APP) while the aminopeptidase N blocker pl250 did not modify fl‐Aβ recovery. In agreement with this observation, we establish that over‐expressed APA drastically reduces, in a calcium dependent manner, fl‐Aβ but not APP IntraCellular Domain in a cell‐free model of Aβ production. In agreement with the above data, we show that recombinant APA degrades fl‐Aβ in a pl302‐sensitive manner. Interestingly, we also show that EC33 and pl302 lower staurosporine‐stimulated activation of caspase‐3 in wild‐type fibroblasts but not in βAPP/β‐amyloid precursor protein‐like protein 2 (APLP2) double knockout fibroblasts, suggesting that protecting endogenous fl‐Aβ physiological production triggers neuroprotective phenotype. By contrast, EC33 does not modify staurosporine‐induced caspase‐3 activation in wild‐type and Swedish‐mutated βAPP‐HEK293 expressing cells that display exacerbated production of Aβ. Overall, our data establish that APA contributes to the N‐terminal truncation of Aβ and suggest that this cleavage is likely abrogating a protective function associated with physiological but not supraphysiological levels of genuine fl‐Aβ peptides.     

Acyl peptide hydrolase, a serine proteinase isolated from conditioned medium of neuroblastoma cells, degrades the amyloid-beta peptide

Considerable evidence indicates that the amyloid-beta (Abeta) peptide, a proteolytic fragment of the amyloid precursor protein, is the pathogenic agent in Alzheimer's disease (AD). A number of proteases have been reported as capable of degrading Abeta, among them: neprilysin, insulin-degrading enzyme, endothelin-converting enzyme-1 and -2, angiotensin-converting enzyme and plasmin. These proteases, originating from a variety of cell types, degrade Abeta of various conformational states and in different cellular locations. We report here the isolation of a serine protease from serum-free conditioned medium of human neuroblastoma cells. Tandem mass spectrometry (MS/MS)-based sequencing of the isolated protein identified acyl peptide hydrolase (APH; EC3.4.19.1) as the active peptidase. APH is one of four members of the prolyl oligopeptidase family of serine proteases expressed in a variety of cells and tissues, including erythrocytes, liver and brain, but its precise biological activity is unknown. Here, we describe the identification of APH as an Abeta-degrading enzyme, and we show that the degradation of Abeta by APH isolated from transfected cells is inhibited by APH-specific inhibitors, as well as by synthetic Abeta peptide. In addition, we cloned APH from human brain and from neuroblastoma cells. Most importantly, our results indicate that APH expression in AD brain is lower than in age-matched controls.     

The SIRT1 activator resveratrol protects SK-N-BE cells from oxidative stress and against toxicity caused by α-synuclein or amyloid-β (1-42) peptide

Human sirtuins are a family of seven conserved proteins (SIRT1-7). The most investigated is the silent mating type information regulation-2 homolog (SIRT1, NM_012238 ), which was associated with neuroprotection in models of polyglutamine toxicity or Alzheimer's disease (AD) and whose activation by the phytocompound resveratrol (RES) has been described. We have examined the neuroprotective role of RES in a cellular model of oxidative stress, a common feature of neurodegeneration. RES prevented toxicity triggered by hydrogen peroxide or 6-hydroxydopamine (6-OHDA). This action was likely mediated by SIRT1 activation, as the protection was lost in the presence of the SIRT1 inhibitor sirtinol and when SIRT1 expression was down-regulated by siRNA approach. RES was also able to protect SK-N-BE from the toxicity arising from two aggregation-prone proteins, the AD-involved amyloid-β (1-42) peptide (Aβ42) and the familiar Parkinson's disease linked α-synuclein(A30P) [α-syn(A30P)]. Alpha-syn(A30P) toxicity was restored by sirtinol addition, while a partial RES protective effect against Aβ42 was found even in presence of sirtinol, thus suggesting a direct RES effect on Aβ42 fibrils. We conclude that SIRT1 activation by RES can prevent in our neuroblastoma model the deleterious effects triggered by oxidative stress or α-syn(A30P) aggregation, while RES displayed a SIRT1-independent protective action against Aβ42.     

Neurotoxic traffic: uncovering the mechanics of amyloid production in Alzheimer's disease

Alzheimer's disease (AD) is thought by many to result from the accumulation of the neurotoxic amyloid-β (Aβ) peptide in brain parenchyma. The process by which Aβ is proteolytically derived from the larger amyloid precursor protein (APP) has been the focus of much attention in the AD research field over the past decade. Recently, several of the proteins directly involved in the generation of Aβ have been identified and characterized providing a number of viable therapeutic targets for the treatment of AD. However, the cellular mechanisms by which these proteins interact in the proteolytic processing of APP have not been well defined, nor are they readily apparent when one considers what is known about the intracellular localization and trafficking of the various participants. This article will review the underlying cell biology of Aβ production and discuss the mechanistic options for APP processing given the current knowledge of the proteases involved.     

Accelerated release of exosome-associated GM1 ganglioside (GM1) by endocytic pathway abnormality: another putative pathway for GM1-induced amyloid fibril formation

Exosomes are extracellularly released small vesicles that are derived from multivesicular bodies formed via the endocytic pathway. We treated pheochromocytoma PC12 cells with chloroquine, an acidotropic agent, which potently perturbs membrane trafficking from endosomes to lysosomes. Chloroquine treatment increased the level of GM1 ganglioside in cell media only when the cells were exposed to KCl for depolarization, which is known to enhance exosome release from neurons. In the sucrose-density-gradient fractionation of cell media, GM1 ganglioside was exclusively recovered with Alix, a specific marker of exosomes, in the fractions with the density corrresponding to that of exosomes. Notably, amyloid-β assembly was markedly accelerated when incubated with the exosome fraction prepared from the culture media of PC12 cells treated with chloroquine and KCl. Furthermore, amyloid-β assembly was significantly suppressed by the co-incubation with an antibody specific to GM1-bound amyloid-β, an endogenous seed for amyloid formation of Alzheimer's disease. Together with our previous finding that chloroquine treatment induces the accumulation of GM1 ganglioside in early endosomes, results of this study suggest that endocytic pathway abnormality accelerates the release of exosome-associated GM1 ganglioside following its accumulation in early endosomes. Furthermore, this study also suggests that extracellular amyloid fibril formation is induced by not only GM1 gangliosides accumulated on the surface of the cells but also those released in association with exosomes.     

The Arctic Alzheimer mutation favors intracellular amyloid-beta production by making amyloid precursor protein less available to alpha-secretase

Mutations within the amyloid-beta (Abeta) domain of the amyloid precursor protein (APP) typically generate hemorrhagic strokes and vascular amyloid angiopathy. In contrast, the Arctic mutation (APP E693G) results in Alzheimer's disease. Little is known about the pathologic mechanisms that result from the Arctic mutation, although increased formation of Abeta protofibrils in vitro and intraneuronal Abeta aggregates in vivo suggest that early steps in the amyloidogenic pathway are facilitated. Here we show that the Arctic mutation favors proamyloidogenic APP processing by increased beta-secretase cleavage, as demonstrated by altered levels of N- and C-terminal APP fragments. Although the Arctic mutation is located close to the alpha-secretase site, APP harboring the Arctic mutation is not an inferior substrate to a disintegrin and metalloprotease-10, a major alpha-secretase. Instead, the localization of Arctic APP is altered, with reduced levels at the cell surface making Arctic APP less available for alpha-secretase cleavage. As a result, the extent and subcellular location of Abeta formation is changed, as revealed by increased Abeta levels, especially at intracellular locations. Our findings suggest that the unique clinical symptomatology and neuropathology associated with the Arctic mutation, but not with other intra-Abeta mutations, could relate to altered APP processing with increased steady-state levels of Arctic Abeta, particularly at intracellular locations.     

Late endocytic dysfunction as a putative cause of amyloid fibril formation in Alzheimer's disease

The assembly of amyloid β-protein to amyloid fibrils is a critical event in Alzheimer's disease. Evidence exists that endocytic pathway abnormalities, including the enlargement of early endosomes, precede the extraneuronal amyloid fibril deposition in the brain. We determined whether endocytic dysfunction potently promotes the assembly of amyloid β-protein on the surface of cultured cells. Blocking the early endocytic pathway by clathrin suppression, inactivation of small GTPases, removal of membrane cholesterol, and Rab5 knockdown did not result in amyloid fibril formation on the cell surface from exogenously added soluble amyloid β-protein. In contrast, blocking the late endocytic pathway by Rab7 suppression markedly induced the amyloid fibril formation in addition to the enlargement of early endosomes. Notably, a monoclonal antibody specific to GM1-ganglioside-bound amyloid β-protein, an endogenous seed for Alzheimer amyloid, completely blocks the amyloid fibril formation. Our results suggest that late but not early endocytic dysfunction contributes to the amyloid fibril formation by facilitating the generation of amyloid seed in the Alzheimer's brain.     

ApoE protects cortical neurones against neurotoxicity induced by the non-fibrillar C-terminal domain of the amyloid-beta peptide

Although the genetic link between the epsilon 4 allele of apolipoprotein E (apoE) and Alzheimer's disease (AD) is well established, the apoE isoform-specific activity underlying this correlation remains unclear. We have recently characterized the interaction of the soluble the amyloid-beta peptide (A beta) with model membrane and demonstrated that non-fibrillar A beta peptide, including N-terminal truncated forms of A beta, induced apoptotic cell death in primary rat cortical neurones in vitro. To further investigate the potential interaction between apoE and A beta in the pathogenesis of AD, we have determined the effect of apoE isoforms on the neurotoxicity of non-fibrillar A beta peptides. We demonstrate here that the apoE2 and E3 isoforms protect cortical neurones against apoptotic cell death induced by a non-fibrillar form of the A beta(1-40), A beta(12-42), A beta(29-40) and A beta(29-42) peptides, whereas apoE4 had no effect. This effect involves the formation of stable complexes between apoE and the C-terminal domain (e.g. amino acids 29-40) of A beta(1-40). Interestingly, apoE had no effect on the toxicity induced by aggregated A beta peptides, suggesting a lack of interaction between apoE and amyloid fibrils. Our results provide evidence that interaction with the C-terminal domain of A beta, apoE2 and E3, but not apoE4, inhibits the interactions of the non-fibrillar A beta peptide with the plasma membrane of neurones, A beta peptide aggregation and subsequent neurotoxicity.     

Neuroprotective effects of donepezil through inhibition of GSK-3 activity in amyloid-β-induced neuronal cell death

Acetylcholinesterase inhibitors (AChE-inhibitors) are used for the treatment of Alzheimer's disease. Recently, the AChE-inhibitor donepezil was found to have neuroprotective effects. However, the protective mechanisms of donepezil have not yet been clearly identified. We investigated the neuroprotective effects of donepezil and other AChE-inhibitors against amyloid-β1–42 (Aβ42)-induced neurotoxicity in rat cortical neurons. To evaluate the neuroprotective effects of AChE-inhibitors, primary cultured cortical neurons were pre-treated with several concentrations of AChE-inhibitors for 24 h and then treated with 20 μM Aβ42 for 6 h. In addition to donepezil, other AChE-inhibitors (galantamine and huperizine A) also showed increased neuronal cell viability against Aβ42 toxicity in a concentration-dependent manner. However, we demonstrated that donepezil has a more potent effect in inhibiting glycogen synthase kinase-3 (GSK-3) activity compared with other AChE-inhibitors. The neuroprotective effects of donepezil were blocked by LY294002 (10 μM), a phosphoinositide 3 kinase inhibitor, but only partially by mecamylamine (10 μM), a blocker of nicotinic acetylcholine receptors. Additionally, donepezil's neuroprotective mechanism was related to the enhanced phosphorylation of Akt and GSK-3β and reduced phosphorylation of tau and glycogen synthase. These results suggest that donepezil prevents Aβ42-induced neurotoxicity through the activation of phosphoinositide 3 kinase/Akt and inhibition of GSK-3, as well as through the activation of nicotinic acetylcholine receptors.     

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.     

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.     

A traditional medicinal herb Paeonia suffruticosa and its active constituent 1,2,3,4,6-penta-O-galloyl-β-d-glucopyranose have potent anti-aggregation effects on Alzheimer's amyloid β proteins in vitro and in vivo

The deposition of amyloid β (Aβ) protein is a consistent pathological hallmark of Alzheimer's disease (AD) brains; therefore, inhibition of Aβ fibril formation and destabilization of pre-formed Aβ fibrils is an attractive therapeutic and preventive strategy in the development of disease-modifying drugs for AD. This study demonstrated that Paeonia suffruticosa , a traditional medicinal herb, not only inhibited fibril formation of both Aβ_1–40 and Aβ_1–42 but it also destabilized pre-formed Aβ fibrils in a concentration-dependent manner. Memory function was examined using the passive-avoidance task followed by measurement of Aβ burden in the brains of Tg2576 transgenic mice. The herb improved long-term memory impairment in the transgenic mice and inhibited the accumulation of Aβ in the brain. Three-dimensional HPLC analysis revealed that a water extract of the herb contained several different chemical compounds including 1,2,3,4,6-penta- O -galloyl-β- d -glucopyranose (PGG). No obvious adverse/toxic were found following treatment with PGG. As was observed with Paeonia suffruticosa , PGG alone inhibited Aβ fibril formation and destabilized pre-formed Aβ fibrils in vitro and in vivo . Our results suggest that both Paeonia suffruticosa and its active constituent PGG have strong inhibitory effects on formation of Aβ fibrils in vitro and in vivo . PGG is likely to be a safe and promising lead compound in the development of disease-modifying drugs to prevent and/or cure AD.     

Proteolytic Degradation of Alzheimer's Disease Amyloid β-Peptide by a Metalloproteinase from Microglia Cells

Abstract: The cerebral deposition of amyloid β-peptide (Aβ) is a histopathological characteristic of Alzheimer's disease. Because an impaired clearance of Aβ might be involved in the disease, we investigated the proteolytic degradation of synthetic Aβ (40-residue peptide) in cultures of glial cells and characterized a protease involved. Whereas rat astrocytes had a very low degradation capacity, cultivated rat microglia cells cleaved Aβ. Microglia activity was considerably enhanced by stimulation with lipopolysaccharide and to a lesser extent by phorbol esters. Most of the Aβ-degrading activity was released into the medium. By use of selective inhibitors the protease was characterized as a metalloprotease of ∼200 kDa that was different from neutral endopeptidase (a neuropeptide-degrading enzyme), matrix metalloproteases, or macrophage elastase. Its activity was efficiently reduced by four hydroxamic acid-based zinc-metalloprotease inhibitors that have been shown to inhibit membrane protein secretases (disintegrins). We conclude that activated microglia cells might impair amyloid plaque formation by release of a metalloprotease that degrades soluble Aβ before polymerization.     

Sulfatides facilitate apolipoprotein E-mediated amyloid-beta peptide clearance through an endocytotic pathway

Amyloid-β (Aβ) accumulation and fibril formation are key pathologic characteristics of Alzheimer's disease (AD). We have previously found that sulfatide depletion occurs at the earliest stages of AD. To further identify the role of sulfatides in the pathogenesis of AD as well as the interactions between apolipoprotein E (apoE), sulfatides, and Aβ peptides, we examined alterations in the clearance of apoE-mediated Aβ peptides after sulfatide supplementation to cell culture systems. We demonstrated that sulfatides markedly facilitate apoE-mediated clearance of Aβ peptides endogenously generated from H4-APPwt cells through an endocytotic pathway. Moreover, we found that the uptake of Aβ42 mediated by sulfatides was selective in comparison to that of Aβ40. We excluded the possibility that the supplementation of sulfatides and/or apoE altered the production of Aβ peptides from H4-APPwt cells through determination of the clearance of Aβ peptides from conditioned H4-APPwt cell media by neuroblastoma cells which do not appreciably generate Aβ peptides. Finally, we demonstrated that the sulfate galactose moiety of sulfatides is essential for the sulfatide-facilitated clearance of Aβ peptides. Collectively, the current study provides insight into a molecular mechanism leading to Aβ clearance/deposition, highlights the significance of sulfatide deficiency at the earliest clinically recognizable stage of AD, and identifies a potential new direction for therapeutic intervention for the disease.     

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.     

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.