The human amyloid-beta precursor protein770 mutation V717F generates peptides longer than amyloid-beta-(40-42) and flocculent amyloid aggregates

One of the familial forms of Alzheimer's disease (AD) encodes the amyloid-beta precursor protein (AbetaPP) substitution mutation V717F. This mutation is relevant to AD research, since it has been utilized to generate transgenic mice models to study AD pathology and therapeutic interventions. Amyloid beta (Abeta) peptides were obtained from the cerebral tissue of three familial AD subjects carrying the AbetaPP V717F mutation. A combination of ultracentrifugation, size-exclusion, and reverse-phase high performance liquid chromatography, tryptic and cyanogen bromide hydrolysis, amino acid analysis, and matrix-assisted laser desorption ionization and surface-enhanced laser desorption ionization mass spectrometry was used to characterize the familial AD mutant Abeta peptides. The AbetaPP V717F mutation, located 4-6 residues beyond the wild-type AbetaPP gamma-secretase cleavage site, yielded longer Abeta peptides with C termini between residues 43 and 54. In the cerebral cortex these peptides aggregated into thin water- and SDS-insoluble amyloid bundles that condensed into flocculent spherical plaques. In the leptomeningeal arteries the amyloid was deposited in moderate amounts and was primarily composed of the shorter and more soluble Abeta species ending at residues 40, 42, and 44. The single V717F mutation in AbetaPP results in distinctive and drastic changes in the length and tertiary structure of Abeta peptides, which appear to be responsible for the earlier clinical manifestations of dementia and death of these patients.     

Reproductive hormones regulate the selective permeability of the blood-brain barrier

Reproductive hormones have been demonstrated to modulate both gap and tight junction protein expression in the ovary and other reproductive tissues, however the effects of changes in reproductive hormones on the selective permeability of the blood-brain barrier (BBB) remain unclear. Age-related declines in BBB integrity correlate with the loss of serum sex steroids and increase in gonadotropins with menopause/andropause. To examine the effect of reproductive senescence on BBB permeability and gap and tight junction protein expression/localization, female mice at 3 months of age were either sham operated (normal serum E2 and gonadotropins), ovariectomized (low serum E2 and high serum gonadotropins) or ovariectomized and treated with the GnRH agonist leuprolide acetate (low serum E2 and gonadotropins). Ovariectomy induced a 2.2-fold increase in Evan's blue dye extravasation into the brain. The expression and localization of the cytoplasmic membrane-associated tight junction protein zona occludens 1 (ZO-1) in microvessels was not altered among groups indicating that the increased paracellular permeability was not due to changes in this tight junction protein. However, ovariectomy induced a redistribution of the gap junction protein connexin-43 (Cx43) such that immunoreactivity relocalized from along the extracellular microvascular endothelium to become associated with endothelial cells. An increase in Cx43 expression in the mouse brain following ovariectomy was suppressed in ovariectomized animals treated with leuprolide acetate, indicating that serum gonadotropins rather than sex steroids were modulating Cx43 expression. These results suggest that elevated serum gonadotropins following reproductive senescence may be one possible cause of the loss of selective permeability of the BBB at this time. Furthermore, these findings implicate Cx43 in mediating changes in BBB permeability, and serum gonadotropins in the cerebropathophysiology of age-related neurodegenerative diseases such as stroke and Alzheimer's disease.     

Neuritic deposits of amyloid-beta peptide in a subpopulation of central nervous system-derived neuronal cells

Our goal is to understand the pathogenesis of amyloid-beta (Abeta) deposition in the Alzheimer's disease (AD) brain. We established a cell culture system where central nervous system-derived neuronal cells (CAD cells) produce and accumulate within their processes large amounts of Abeta peptide, similar to what is believed to occur in brain neurons, in the initial phases of AD. Using this system, we show that accumulation of Abeta begins within neurites, prior to any detectable signs of neurodegeneration or abnormal vesicular transport. Neuritic accumulation of Abeta is restricted to a small population of neighboring cells that express normal levels of amyloid-beta precursor protein (APP) but show redistribution of BACE1 to the processes, where it colocalizes with Abeta and markers of late endosomes. Consistently, cells that accumulate Abeta appear in isolated islets, suggesting their clonal origin from a few cells that show a propensity to accumulate Abeta. These results suggest that Abeta accumulation is initiated in a small number of neurons by intracellular determinants that alter APP metabolism and lead to Abeta deposition and neurodegeneration. CAD cells appear to recapitulate the biochemical processes leading to Abeta deposition, thus providing an experimental in vitro system for studying the molecular pathobiology of AD.     

The endocrine dyscrasia that accompanies menopause and andropause induces aberrant cell cycle signaling that triggers re-entry of post-mitotic neurons into the cell cycle,neurodysfunction, neurodegeneration and cognitive disease

This article is part of a Special Issue “SBN 2014”.Sex hormones are physiological factors that promote neurogenesis during embryonic and fetal development. During childhood and adulthood these hormones support the maintenance of brain structure and function via neurogenesis and the formation of dendritic spines, axons and synapses required for the capture, processing and retrieval of information (memories). Not surprisingly, changes in these reproductive hormones that occur with menopause and during andropause are strongly correlated with neurodegeneration and cognitive decline. In this connection, much evidence now indicates that Alzheimer's disease (AD) involves aberrant re-entry of post-mitotic neurons into the cell cycle. Cell cycle abnormalities appear very early in the disease, prior to the appearance of plaques and tangles, and explain the biochemical, neuropathological and cognitive changes observed with disease progression. Intriguingly, a recent animal study has demonstrated that induction of adult neurogenesis results in the loss of previously encoded memories while decreasing neurogenesis after memory formation during infancy mitigated forgetting. Here we review the biochemical, epidemiological and clinical evidence that alterations in sex hormone signaling associated with menopause and andropause drive the aberrant re-entry of post-mitotic neurons into an abortive cell cycle that leads to neurite retraction, neuron dysfunction and neuron death. When the reproductive axis is in balance, gonadotropins such as luteinizing hormone (LH), and its fetal homolog, human chorionic gonadotropin (hCG), promote pluripotent human and totipotent murine embryonic stem cell and neuron proliferation. However, strong evidence supports menopausal/andropausal elevations in the LH:sex steroid ratio as driving aberrant mitotic events. These include the upregulation of tumor necrosis factor; amyloid-β precursor protein processing towards the production of mitogenic Aβ; and the activation of Cdk5, a key regulator of cell cycle progression and tau phosphorylation (a cardinal feature of both neurogenesis and neurodegeneration). Cognitive and biochemical studies confirm the negative consequences of a high LH:sex steroid ratio on dendritic spine density and human cognitive performance. Prospective epidemiological and clinical evidence in humans supports the premise that rebalancing the ratio of circulating gonadotropins:sex steroids reduces the incidence of AD. Together, these data support endocrine dyscrasia and the subsequent loss of cell cycle control as an important etiological event in the development of neurodegenerative diseases including AD, stroke and Parkinson's disease.     

Sialylation enhances the secretion of neurotoxic amyloid-beta peptides

Alzheimer's disease (AD) is characterized by amyloid-beta peptide (Abeta) deposition in the brain. Abeta is produced by sequential cleavage of amyloid precursor protein (APP) by beta-secretase (BACE1: beta-site APP-cleaving enzyme 1) and gamma-secretase. Previously, we demonstrated that BACE1 also cleaves beta-galactoside alpha2,6-sialyltransferase (ST6Gal-I) and down-regulates its transferase activity. Here, we report that overexpression of ST6Gal-I in Neuro2a cells enhanced alpha2,6-sialylation of endogenous APP and increased the extracellular levels of its metabolites [Abeta by two-fold, soluble APPbeta (sAPPbeta) by three-fold and sAPPalpha by 2.5-fold). Sialylation-deficient mutant (Lec-2) cells secreted half as much Abeta as wild-type Chinese hamster ovary (CHO) cells. Furthermore, wild-type CHO cells showed enhanced secretion of the APP metabolites upon ST6Gal-I overexpression, whereas Lec-2 cells did not, indicating that the secretion enhancement requires sialylation of cellular protein(s). Secretion of metabolites from a mutant APP (APP-Asn467,496Ala) that lacked N-glycosylation sites was not enhanced upon ST6Gal-I overexpression, suggesting that the N-glycans on APP itself are required for the enhanced secretion. In the mouse brain, the amount of alpha2,6-sialylated APP appeared to be correlated with the sAPPbeta level. These results suggest that sialylation of APP promotes its metabolic turnover and could affect the pathology of AD.     

Alzheimer vaccine: amyloid-beta on trial

A new therapeutic approach is being developed for the treatment of Alzheimer's disease (AD). This approach involves the deliberate induction of an autoimmune response to amyloid-beta (Abeta) peptide, the constituent of neuritic plaques that is thought to cause the neurodegeneration and dementia in AD. If this approach is to be effective, antibodies must be produced that can selectively target the toxic forms of Abeta, while leaving the functionally-relevant forms of Abeta and its precursor protein untouched. Furthermore, an approach needs to be found that avoids provoking an acute neuroinflammatory response. The situation is made even more challenging by uncertainty regarding which isoforms of Abeta contribute to the pathogenesis of AD.     

Microglial transplantation increases amyloid-beta clearance in Alzheimer model rats

Immunization with amyloid-beta (Abeta) peptides, a therapeutic approach in Alzheimer's disease (AD), reduces brain Abeta, and microglial Abeta phagocytosis has been proposed as an Abeta-lowering mechanism. We transplanted rat microglia into the rat lateral ventricle just after intra-hippocampal Abeta injection, and then investigated the contribution of exogenous microglia to Abeta clearance. Migration of exogenous microglia from the lateral ventricle to Abeta plaque was detected by magnetic resonance imaging and histochemical analysis, and the clearance of Abeta was increased by transplantation. These results suggest the possible usefulness of exogenous microglia to the therapeutic approach in AD.     

Apolipoprotein E enhances uptake of soluble but not aggregated amyloid-beta protein into synaptic terminals

The cellular mechanism by which apolipoprotein E (apoE) affects the pathogenesis of Alzheimer's disease (AD) is not understood. We have examined the effect of apolipoprotein E on the internalization of exogenous amyloid-beta 1-40 (Abeta40) into a rat brain crude synaptosomal preparation. Abeta40 peptide in soluble (within 1 h of dilution in buffer) or aggregated (aged 4 days before dilution in buffer) form was pre-incubated with lipidated apoE then added to synaptosomes; intraterminal amyloid-beta labeling was quantified using flow cytometry following immunolabeling with the anti-Abeta (10G4) antibody. The number of Abeta-positive synaptosomes was increased ( approximately 50%) by treatment with a soluble Abeta/apoE mixture compared with treatment with soluble Abeta40 alone. However, when the Abeta was aggregated, less sodium dodecyl sulfate (SDS)-stable Abeta/apoE complex was formed and the addition of apoE decreased the number of Abeta-positive terminals. The addition of the lipoprotein-receptor related protein (LRP) antagonist receptor-associated protein (RAP) inhibited the apoE-induced increase in synaptosomal Abeta, and controls treated with trypsin and heparinase confirm intraterminal localization of the majority of the soluble Abeta. The apoE-mediated increase in Abeta labeling was confirmed in intact cells by immunocytochemistry of dorsal root ganglion (DRG) neurons. These results suggest that complex formation with apoE enhances internalization of soluble Abeta uptake into terminals.     

Adult mouse astrocytes degrade amyloid-beta in vitro and in situ

Alzheimer disease (AD) is a progressive neurodegenerative disorder characterized by excessive deposition of amyloid-beta (Abeta) peptides in the brain. One of the earliest neuropathological changes in AD is the accumulation of astrocytes at sites of Abeta deposition, but the cause or significance of this cellular response is unclear. Here we show that cultured adult mouse astrocytes migrate in response to monocyte chemoattractant protein-1 (MCP-1), a chemokine present in AD lesions, and cease migration upon interaction with immobilized Abeta(1-42). We also show that astrocytes bind and degrade Abeta(1-42). Astrocytes plated on Abeta-laden brain sections from a mouse model of AD associate with the Abeta deposits and reduce overall Abeta levels in these sections. Our results suggest a novel mechanism for the accumulation of astrocytes around Abeta deposits, indicate a direct role for astrocytes in degradation of Abeta and implicate deficits in astroglial clearance of Abeta in the pathogenesis of AD. Treatments that increase removal of Abeta by astrocytes may therefore be a critical mechanism to reduce the neurodegeneration associated with AD.     

Targeted proteomics in Alzheimer's disease: focus on amyloid-beta

Diagnosis and monitoring of sporadic Alzheimer's disease (AD) have long depended on clinical examination of individuals with end-stage disease. However, upcoming anti-AD therapies are optimally initiated when individuals show very mild signs of neurodegeneration. There is a developing consensus for cerebrospinal fluid amyloid-beta (Abeta) as a core biomarker for the mild cognitive impairment stage of AD. Abeta is directly involved in the pathogenesis of AD or tightly correlated with other primary pathogenic factors. It is produced from amyloid precursor protein (APP) by proteolytic processing that depends on the beta-site APP-cleaving enzyme 1 and the gamma-secretase complex, and is degraded by a broad range of proteases. This review summarizes targeted proteomic studies of Abeta in biological fluids and identifies clinically useful markers of disrupted Abeta homeostasis in AD. The next 5 years will see a range of novel assays developed on the basis of these results. From a longer perspective, establishment of the most effective combinations of different biomarkers and other diagnostic modalities may be foreseen.     

Papillomavirus-like particles are an effective platform for amyloid-beta immunization in rabbits and transgenic mice

Immunization with amyloid-beta (Abeta) prevents the deposition of Abeta in the brain and memory deficits in transgenic mouse models of Alzheimer's disease (AD), opening the possibility for immunotherapy of AD in humans. Unfortunately, the first human trial of Abeta vaccination was complicated, in a small number of vaccinees, by cell-mediated meningoencephalitis. To develop an Abeta vaccine that lacks the potential to induce autoimmune encephalitis, we have generated papillomavirus-like particles (VLP) that display 1-9 aa of Abeta protein repetitively on the viral capsid surface (Abeta-VLP). This Abeta peptide was chosen because it contains a functional B cell epitope, but lacks known T cell epitopes. Rabbit and mouse vaccinations with Abeta-VLP were well tolerated and induced high-titer autoAb against Abeta, that inhibited effectively assembly of Abeta(1-42) peptides into neurotoxic fibrils in vitro. Following Abeta-VLP immunizations of APP/presenilin 1 transgenic mice, a model for human AD, we observed trends for reduced Abeta deposits in the brain and increased numbers of activated microglia. Furthermore, Abeta-VLP vaccinated mice also showed increased levels of Abeta in plasma, suggesting efflux from the brain into the vascular compartment. These results indicate that the Abeta-VLP vaccine induces an effective humoral immune response to Abeta and may thus form a basis to develop a safe and efficient immunotherapy for human AD.     

Role of high mobility group protein-1 (HMG1) in amyloid-beta homeostasis

In Alzheimer's disease (AD), fibrillar amyloid-beta (Abeta) peptides form senile plaques associated with activated microglia. Recent studies have indicated that microglial Abeta clearance is facilitated by several activators such as transforming growth factor-beta1 (TGF-beta1). The relationship between microglia and Abeta formation and deposition is still unclear. In the present study, high mobility group protein-1 (HMG1) inhibited the microglial uptake of Abeta (1-42) in the presence and absence of TGF-beta1. In addition, HMG1 bound to Abeta (1-42) and stabilized the oligomerization. In AD brains, protein levels of HMG1 were significantly increased in both the cytosolic and particulate fractions, and HMG1 and Abeta were colocalized in senile plaques associated with microglia. These results suggest that HMG1 may regulate the homeostasis of extracellular Abeta (1-42) and Abeta oligomerization.     

Secretion and intracellular generation of truncated Abeta in beta-site amyloid-beta precursor protein-cleaving enzyme expressing human neurons

Insoluble pools of the amyloid-beta peptide (Abeta) in brains of Alzheimer's disease patients exhibit considerable N- and C-terminal heterogeneity. Mounting evidence suggests that both C-terminal extensions and N-terminal truncations help precipitate amyloid plaque formation. Although mechanisms underlying the increased generation of C-terminally extended peptides have been extensively studied, relatively little is known about the cellular mechanisms underlying production of N-terminally truncated Abeta. Thus, we used human NT2N neurons to investigate the production of Abeta11-40/42 from amyloid-beta precursor protein (APP) by beta-site APP-cleaving enzyme (BACE). When comparing undifferentiated human embryonal carcinoma NT2- cells and differentiated NT2N neurons, the secretion of sAPP and Abeta correlated with BACE expression. To study the effects of BACE expression on endogenous APP metabolism in human cells, we overexpressed BACE in undifferentiated NT2- cells and NT2N neurons. Whereas NT2N neurons produced both full-length and truncated Abeta as a result of normal processing of endogenous APP, BACE overexpression increased the secretion of Abeta1-40/42 and Abeta11-40/42 in both NT2- cells and NT2N neurons. Furthermore, BACE overexpression resulted in increased intracellular Abeta1-40/42 and Abeta11-40/42. Therefore, we conclude that Abeta11-40/42 is generated prior to deposition in senile plaques and that N-terminally truncated Abeta peptides may contribute to the downstream effects of amyloid accumulation in Alzheimer's disease.     

Cytosolic amyloid-beta peptide 42 escaping from degradation induces cell death

Accumulating evidence suggests that intracellular amyloid-beta (Abeta) peptide triggers the early pathological events in Alzheimer's disease (AD). However, little is known about the consequence of cytosolic Abeta. In this study, we ectopically expressed Abeta42 in the cytoplasm of SH-SY5Y neuroblastoma cells by expressing a fusion protein of GFP-tagged ubiquitin and Abeta42 (GFPUb-Abeta42). Although GFPUb and Abeta42 are stochastically produced with the same molar ratio in the cytoplasm, Abeta42 was completely degraded in more than 50% of the GFPUb-expressing cells. However, if Abeta42 was not degraded in their cytoplasm, then Abeta42-expressing cells underwent apoptosis. The number of Abeta42-expressing cells is significantly increased by the inhibition of proteasome with MG132. Cytosolic Abeta42 which has escaped degradation inhibits proteasome and thereby may accelerate the accumulation of Abeta42 and its detrimental effects. Our findings suggest that cells have the potential to degrade Abeta42 in their cytoplasm but if Abeta42 appears in the cytoplasm due to its incomplete degradation, it accumulates and may trigger the fatal cascade of pathology of AD.     

Isoform-specific interactions of human apolipoprotein E to an intermediate conformation of human Alzheimer amyloid-beta peptide

Brain plaque deposits of amyloid-beta peptide (Abeta) is a pathological hallmark of Alzheimer's disease (AD) and apolipoprotein E (apoE) is thought to be involved in its deposition. One hypothesis for the role of apoE in the pathogenesis of AD is that apoE may be involved in deposition or clearance of Abeta by direct protein-to-protein interaction. Lipidated apoE4 bound preferentially to an intermediate aggregated form of Abeta and formed two- to three-fold more binding complexes than isoforms apoE2 or apoE3. The interaction was detected by a sandwich ELISA with capture antibodies specific for the N-terminus of apoE, whereas the interaction was not recognized with a C-terminal antibody. The observations indicate that the C-terminus of apoE4 interacts with the intermediate form of Abeta. The differential risk of AD related to apoE genotype may be the result of an enhanced capacity of apoE4 binding to an intermediate aggregated form of Abeta.     

Conformation-sensitive antibodies against alzheimer amyloid-beta by immunization with a thioredoxin-constrained B-cell epitope peptide

Immunotherapy against the amyloid-beta (Abeta) peptide is a valuable potential treatment for Alzheimer disease (AD). An ideal antigen should be soluble and nontoxic, avoid the C-terminally located T-cell epitope of Abeta, and yet be capable of eliciting antibodies that recognize Abeta fibrils and neurotoxic Abeta oligomers but not the physiological monomeric species of Abeta. We have described here the construction and immunological characterization of a recombinant antigen with these features obtained by tandem multimerization of the immunodominant B-cell epitope peptide Abeta1-15 (Abeta15) within the active site loop of bacterial thioredoxin (Trx). Chimeric Trx(Abeta15)n polypeptides bearing one, four, or eight copies of Abeta15 were constructed and injected into mice in combination with alum, an adjuvant approved for human use. All three polypeptides were found to be immunogenic, yet eliciting antibodies with distinct recognition specificities. The anti-Trx(Abeta15)4 antibody, in particular, recognized Abeta42 fibrils and oligomers but not monomers and exhibited the same kind of conformational selectivity against transthyretin, an amyloidogenic protein unrelated in sequence to Abeta. We have also demonstrated that anti-Trx(Abeta15)4, which binds to human AD plaques, markedly reduces Abeta pathology in transgenic AD mice. The data indicate that a conformational epitope shared by oligomers and fibrils can be mimicked by a thioredoxin-constrained Abeta fragment repeat and identify Trx(Abeta15)4 as a promising new tool for AD immunotherapy.     

Amyloid precursor protein and amyloid beta peptide in human platelets. Role of cyclooxygenase and protein kinase C

The main component of Alzheimer's disease (AD) senile plaques is amyloid-beta peptide (Abeta), a proteolytic fragment of the amyloid precursor protein (APP). Platelets contain both APP and Abeta and may contribute to the perivascular amyloid deposition seen in AD. However, no data are available concerning the biochemical mechanism(s) involved in their formation and release by these cells. We found that human platelets released APP and Abeta following activation with collagen or arachidonic acid. Inhibition of platelet cyclooxygenase (COX) reduced APP but not Abeta release following those stimuli. In contrast, activation of platelets by thrombin and calcium ionophore caused release of both APP and Abeta in a COX-independent fashion. Ex vivo studies showed that, despite suppression of COX activity, administration of aspirin did not modify Abeta or APP levels in serum or plasma, suggesting that this enzyme plays only a minor role in vivo. We examined the regulation of APP cleavage and release from activated platelets and found that cleavage requires protein kinase C (PKC) activity and is regulated by the intracellular second messengers phosphatidylinositol 2-phosphate and Ca(2+). Our data provide the first evidence that in human platelets COX is a minor component of APP secretion whereas PKC plays a major role in the secretory cleavage of APP. By contrast, Abeta release may represent secretion of preformed peptide and is totally independent of both COX and PKC activity.     

Docosahexaenoic acid prevents neuronal apoptosis induced by soluble amyloid-beta oligomers

A growing body of evidence supports the notion that soluble oligomers of amyloid-beta (Abeta) peptide interact with the neuronal plasma membrane, leading to cell injury and inducing death-signalling pathways that could account for the increased neurodegeneration occurring in Alzheimer's disease (AD). Docosahexaenoic acid (DHA, C22:6, n-3) is an essential polyunsaturated fatty acid in the CNS and has been shown in several epidemiological and in vivo studies to have protective effects against AD and cognitive alterations. However, the molecular mechanisms involved remain unknown. We hypothesized that DHA enrichment of plasma membranes could protect neurones from apoptosis induced by soluble Abeta oligomers. DHA pre-treatment was observed to significantly increase neuronal survival upon Abeta treatment by preventing cytoskeleton perturbations, caspase activation and apoptosis, as well as by promoting extracellular signal-related kinase (ERK)-related survival pathways. These data suggest that DHA enrichment probably induces changes in neuronal membrane properties with functional outcomes, thereby increasing protection from soluble Abeta oligomers. Such neuroprotective effects could be of major interest in the prevention of AD and other neurodegenerative diseases.     

Metal swap between Zn7-metallothionein-3 and amyloid-beta-Cu protects against amyloid-beta toxicity

Aberrant interactions of copper and zinc ions with the amyloid-beta peptide (Abeta) potentiate Alzheimer's disease (AD) by participating in the aggregation process of Abeta and in the generation of reactive oxygen species (ROS). The ROS production and the neurotoxicity of Abeta are associated with copper binding. Metallothionein-3 (Zn(7)MT-3), an intra- and extracellularly occurring metalloprotein, is highly expressed in the brain and downregulated in AD. This protein protects, by an unknown mechanism, cultured neurons from the toxicity of Abeta. Here, we show that a metal swap between Zn(7)MT-3 and soluble and aggregated Abeta(1-40)-Cu(II) abolishes the ROS production and the related cellular toxicity. In this process, copper is reduced by the protein thiolates forming Cu(I)(4)Zn(4)MT-3, in which an air-stable Cu(I)(4)-thiolate cluster and two disulfide bonds are present. The discovered protective effect of Zn(7)MT-3 from the copper-mediated Abeta(1-40) toxicity may lead to new therapeutic strategies for treating AD.