Immunological aspects of microglia: relevance to Alzheimer's disease

Alzheimer's disease (AD) is a progressive dementing neurologic illness, and the most frequent cause of dementia in the elderly. Neuritic plaques are one of the main neuropathological findings in AD, and the major protein component is the β-amyloid protein (Aβ). Another striking feature of neuritic plaques is the presence of activated microglia, cytokines, and complement components, suggestive of “inflammatory foci” within AD brain. In this review, we will examine the mechanisms by which microglia become activated in AD, emphasizing the role in the Aβ protein and proinflammatory cytokines. As well, pathways for suppression of microglial activation by immunosuppressive cytokines will be described. Inflammation mediated by activated microglia is an important component of AD pathophysiology, and strategies to control this response could provide new therapeutic approaches for the treatment of AD.     

Xanthene food dye, as a modulator of Alzheimer's disease amyloid-beta peptide aggregation and the associated impaired neuronal cell function

Background

Alzheimer''s disease (AD) is the most common form of dementia. AD is a degenerative brain disorder that causes problems with memory, thinking and behavior. It has been suggested that aggregation of amyloid-beta peptide (Aβ) is closely linked to the development of AD pathology. In the search for safe, effective modulators, we evaluated the modulating capabilities of erythrosine B (ER), a Food and Drug Administration (FDA)-approved red food dye, on Aβ aggregation and Aβ-associated impaired neuronal cell function.

Methodology/Principal Findings

In order to evaluate the modulating ability of ER on Aβ aggregation, we employed transmission electron microscopy (TEM), thioflavin T (ThT) fluorescence assay, and immunoassays using Aβ-specific antibodies. TEM images and ThT fluorescence of Aβ samples indicate that protofibrils are predominantly generated and persist for at least 3 days. The average length of the ER-induced protofibrils is inversely proportional to the concentration of ER above the stoichiometric concentration of Aβ monomers. Immunoassay results using Aβ-specific antibodies suggest that ER binds to the N-terminus of Aβ and inhibits amyloid fibril formation. In order to evaluate Aβ-associated toxicity we determined the reducing activity of SH-SY5Y neuroblastoma cells treated with Aβ aggregates formed in the absence or in the presence of ER. As the concentration of ER increased above the stoichiometric concentration of Aβ, cellular reducing activity increased and Aβ-associated reducing activity loss was negligible at 500 µM ER.

Conclusions/Significance

Our findings show that ER is a novel modulator of Aβ aggregation and reduces Aβ-associated impaired cell function. Our findings also suggest that xanthene dye can be a new type of small molecule modulator of Aβ aggregation. With demonstrated safety profiles and blood-brain permeability, ER represents a particularly attractive aggregation modulator for amyloidogenic proteins associated with neurodegenerative diseases.     

Functional role of TGF beta in Alzheimer's disease microvascular injury: lessons from transgenic mice

Recent studies have implicated pro- and anti-inflammatory cytokines as integral to Alzheimer's disease (AD) pathogenesis. Among them, transforming growth factor-β (TGF-β) is emerging as an important factor in regulating inflammatory responses. This multifunctional cytokine might be centrally involved in several aspects of AD pathogenesis by regulating β-amyloid precursor protein synthesis and processing, plaque formation, astroglial and microglial response and neuronal cell death. Among all of these potential roles, studies in transgenic and infusion animal models have shown that TGF-β may primarily contribute to AD pathogenesis by influencing Aβ production and deposition, which in turn might result in damage to the brain microvasculature. The lessons learned from these models are of great interest not only for understanding of the role of TGF-β in AD, but also for future treatments where testing of anti-inflammatory agents such as ibuprofen and an amyloid vaccine hold great promise. In this regard, further elucidation of the signal pathways by which TGF-β exerts its effect in AD might lead to specific targets for further therapeutic intervention.     

Alzheimer Aβ peptide interactions with lipid membranes: Fibrils,oligomers and polymorphic amyloid channels

Fibrillar aggregates of misfolded amyloid proteins are involved in a variety of diseases such as Alzheimer disease (AD), type 2 diabetes, Parkinson, Huntington and prion-related diseases. In the case of AD amyloid β (Aβ) peptides, the toxicity of amyloid oligomers and larger fibrillar aggregates is related to perturbing the biological function of the adjacent cellular membrane. We used atomistic molecular dynamics (MD) simulations of Aβ_9–40 fibrillar oligomers modeled as protofilament segments, including lipid bilayers and explicit water molecules, to probe the first steps in the mechanism of Aβ-membrane interactions. Our study identified the electrostatic interaction between charged peptide residues and the lipid headgroups as the principal driving force that can modulate the further penetration of the C-termini of amyloid fibrils or fibrillar oligomers into the hydrophobic region of lipid membranes. These findings advance our understanding of the detailed molecular mechanisms and the effects related to Aβ-membrane interactions, and suggest a polymorphic structural character of amyloid ion channels embedded in lipid bilayers. While inter-peptide hydrogen bonds leading to the formation of β-strands may still play a stabilizing role in amyloid channel structures, these may also present a significant helical content in peptide regions (e.g., termini) that are subject to direct interactions with lipids rather than with neighboring Aβ peptides.     

Amyloid β protein activates PKC-δ and induces translocation of myristoylated alanine-rich C kinase substrate (MARCKS) in microglia

The increased accumulation of activated microglia containing amyloid β protein (Aβ) around senile plaques is a common pathological feature in subjects with Alzheimer's disease (AD). Much less is known, however, of intracellular signal transduction pathways for microglial activation in response to Aβ. We investigated intracellular signaling in response to Aβ stimulation in primary cultured rat microglia. We found that the kinase activity of PKC-δ but not that of PKC- or - is increased by stimulation of microglia with Aβ, with a striking tyrosine phosphorylation of PKC-δ. In microglia stimulated with Aβ, tyrosine phosphorylation of PKC-δ was evident at the membrane fraction without an overt translocation of PKC-δ. PKC-δ co-immunoprecipitated with MARCKS from microglia stimulated with Aβ. Aβ induced translocation of MARCKS from the membrane fraction to the cytosolic fraction. Immunocytochemical analysis revealed that phosphorylated MARCKS accumulated in the cytoplasm, particularly at the perinuclear region in microglia treated with Aβ. Taken together with our previous observations that Aβ-induced phosphorylation of MARCKS and chemotaxis of microglia are inhibited by either tyrosine kinase or PKC inhibitors, our results provide evidence that Aβ induces phosphorylation and translocation of MARCKS through the tyrosine kinase-PKC-δ signaling pathway in microglia.     

A��42-to-A��40- and Angiotensin-converting Activities in Different Domains of Angiotensin-converting Enzyme

Amyloid β-protein 1–42 (Aβ42) is believed to play a causative role in the development of Alzheimer disease (AD), although it is a minor part of Aβ. In contrast, Aβ40 is the predominant secreted form of Aβ and recent studies have suggested that Aβ40 has neuroprotective effects and inhibits amyloid deposition. We have reported that angiotensin-converting enzyme (ACE) converts Aβ42 to Aβ40, and its inhibition enhances brain Aβ42 deposition (Zou, K., Yamaguchi, H., Akatsu, H., Sakamoto, T., Ko, M., Mizoguchi, K., Gong, J. S., Yu, W., Yamamoto, T., Kosaka, K., Yanagisawa, K., and Michikawa, M. (2007) J. Neurosci. 27, 8628–8635). ACE has two homologous domains, each having a functional active site. In the present study, we identified the domain of ACE, which is responsible for converting Aβ42 to Aβ40. Interestingly, Aβ42-to-Aβ40-converting activity is solely found in the N-domain of ACE and the angiotensin-converting activity is found predominantly in the C-domain of ACE. We also found that the N-linked glycosylation is essential for both Aβ42-to-Aβ40- and angiotensin-converting activities and that unglycosylated ACE rapidly degraded. The domain-specific converting activity of ACE suggests that ACE inhibitors could be designed to specifically target the angiotensin-converting C-domain, without inhibiting the Aβ42-to-Aβ40-converting activity of ACE or increasing neurotoxic Aβ42.     

Alzheimer's Disease Related Markers,Cellular Toxicity and Behavioral Deficits Induced Six Weeks after Oligomeric Amyloid-β Peptide Injection in Rats

Alzheimer’s disease (AD) is a neurodegenerative pathology associated with aging characterized by the presence of senile plaques and neurofibrillary tangles that finally result in synaptic and neuronal loss. The major component of senile plaques is an amyloid-β protein (Aβ). Recently, we characterized the effects of a single intracerebroventricular (icv) injection of Aβ fragment (25–35) oligomers (oAβ_25–35) for up to 3 weeks in rats and established a clear parallel with numerous relevant signs of AD. To clarify the long-term effects of oAβ_25–35 and its potential role in the pathogenesis of AD, we determined its physiological, behavioral, biochemical and morphological impacts 6 weeks after injection in rats. oAβ_25–35 was still present in the brain after 6 weeks. oAβ_25–35 injection did not affect general activity and temperature rhythms after 6 weeks, but decreased body weight, induced short- and long-term memory impairments, increased corticosterone plasma levels, brain oxidative (lipid peroxidation), mitochondrial (caspase-9 levels) and reticulum stress (caspase-12 levels), astroglial and microglial activation. It provoked cholinergic neuron loss and decreased brain-derived neurotrophic factor levels. It induced cell loss in the hippocampic CA subdivisions and decreased hippocampic neurogenesis. Moreover, oAβ_25–35 injection resulted in increased APP expression, Aβ_1–42 generation, and increased Tau phosphorylation. In conclusion, this in vivo study evidenced that the soluble oligomeric forms of short fragments of Aβ, endogenously identified in AD patient brains, not only provoked long-lasting pathological alterations comparable to the human disease, but may also directly contribute to the progressive increase in amyloid load and Tau pathology, involved in the AD physiopathology.     

The Large Hydrophilic Loop of Presenilin 1 Is Important for Regulating ��-Secretase Complex Assembly and Dictating the Amyloid �� Peptide (A��) Profile without Affecting Notch Processing

γ-Secretase is an enzyme complex that mediates both Notch signaling and β-amyloid precursor protein (APP) processing, resulting in the generation of Notch intracellular domain, APP intracellular domain, and the amyloid β peptide (Aβ), the latter playing a central role in Alzheimer disease (AD). By a hitherto undefined mechanism, the activity of γ-secretase gives rise to Aβ peptides of different lengths, where Aβ42 is considered to play a particular role in AD. In this study we have examined the role of the large hydrophilic loop (amino acids 320–374, encoded by exon 10) of presenilin 1 (PS1), the catalytic subunit of γ-secretase, for γ-secretase complex formation and activity on Notch and APP processing. Deletion of exon 10 resulted in impaired PS1 endoproteolysis, γ-secretase complex formation, and had a differential effect on Aβ-peptide production. Although the production of Aβ38, Aβ39, and Aβ40 was severely impaired, the effect on Aβ42 was affected to a lesser extent, implying that the production of the AD-related Aβ42 peptide is separate from the production of the Aβ38, Aβ39, and Aβ40 peptides. Interestingly, formation of the intracellular domains of both APP and Notch was intact, implying a differential cleavage activity between the ϵ/S3 and γ sites. The most C-terminal amino acids of the hydrophilic loop were important for regulating APP processing. In summary, the large hydrophilic loop of PS1 appears to differentially regulate the relative production of different Aβ peptides without affecting Notch processing, two parameters of significance when considering γ-secretase as a target for pharmaceutical intervention in AD.     

Apolipoprotein E and apolipoprotein E receptors modulate A beta-induced glial neuroinflammatory responses

Large numbers of activated glia are a common pathological feature of many neurodegenerative disorders, including Alzheimer's disease (AD). Several different stimuli, including lipopolysaccharide (LPS), dibutyryl (db)cAMP, and aged amyloid-β 1–42 (Aβ), can induce glial activation in vitro, as measured by morphological changes and the production of pro-inflammatory cytokines and oxidative stress molecules. Only Aβ-induced activation is attenuated by the addition of exogenous apolipoprotein E (apoE)-containing particles. In addition, only Aβ also induces an increase in the amount of endogenous apoE, the primary apolipoprotein expressed by astrocytes in the brain. The functional significance of the increase in apoE appears to be to limit the inflammatory response. Indeed, compared to wild type mice, glial cells cultured from apoE knockout mice exhibit an enhanced production of several pro-inflammatory markers in response to treatment with Aβ and other activating stimuli. The mechanism for both the Aβ-induced glial activation and the increase in apoE appears to involve apoE receptors, a variety of which are expressed by both neurons and glia. Experiments using receptor associated protein (RAP), an inhibitor of apoE receptors with a differential affinity for the low-density lipoprotein receptor (LDLR) and the LDLR-related protein (LRP), revealed that LRP mediates Aβ-induced glial activation, while LDLR mediates the Aβ-induced changes in apoE levels. In summary, both an apoE receptor agonist (apoE) and an antagonist (RAP) inhibit Aβ-induced glial cell activation. Thus, apoE receptors appear to translate the presence of extracellular Aβ into cellular responses, both initiating glial cell activation and limiting its scope by inducing apoE, an anti-inflammatory agent.     

Structural Correlates of Antibodies Associated with Acute Reversal of Amyloid ��-related Behavioral Deficits in a Mouse Model of Alzheimer Disease

Immunotherapy targeting of amyloid β (Aβ) peptide in transgenic mouse models of Alzheimer disease (AD) has been widely demonstrated to resolve amyloid deposition as well as associated neuronal, glial, and inflammatory pathologies. These successes have provided the basis for ongoing clinical trials of immunotherapy for treatment of AD in humans. Acute as well as chronic Aβ-targeted immunotherapy has also been demonstrated to reverse Aβ-related behavioral deficits assessing memory in AD transgenic mouse models. We observe that three antibodies targeting the same linear epitope of Aβ, Aβ_3–7, differ in their ability to reverse contextual fear deficits in Tg2576 mice in an acute testing paradigm. Reversal of contextual fear deficit by the antibodies does not correlate with in vitro recognition of Aβ in a consistent or correlative manner. To better define differences in antigen recognition at the atomic level, we determined crystal structures of Fab fragments in complex with Aβ. The conformation of the Aβ peptide recognized by all three antibodies was highly related and is also remarkably similar to that observed in independently reported Aβ:antibody crystal structures. Sequence and structural differences between the antibodies, particularly in CDR3 of the heavy chain variable region, are proposed to account for differing in vivo properties of the antibodies under study. These findings provide a structural basis for immunotherapeutic strategies targeting Aβ species postulated to underlie cognitive deficits in AD.     

Estrogen (E2) and glucocorticoid (Gc) effects on microglia and A beta clearance in vitro and in vivo

The accumulation of fibrillar aggregates of beta Amyloid (Aβ) in Alzheimer's Disease (AD) brain is associated with chronic brain inflammation. Although activated microglia (μglia) can potentially clear toxic amyloid, chronic activation may lead to excessive production of neurotoxins. Recent epidemiological and clinical data have raised questions about the use of anti-inflammatory steroids (glucocorticoids, Gcs) and estrogens for treatment or prevention of AD. Since very little is known about steroid effects on μglial interactions with amyloid, we investigated the effects of the synthetic Gc dexamethasone (DXM) and 17-β estradiol (E2) in vitro in a murine μglial-like N9 cell line on toxin production and intracellular Aβ accumulation. To determine whether the steroid alterations of Aβ uptake in vitro had relevance in vivo, we examined the effects of these steroids on Aβ accumulation and μglial responses to Aβ infused into rat brain. Our in vitro data demonstrate for the first time that Gc dose-dependently enhanced μglial Aβ accumulation and support previous work showing that E2 enhances Aβ uptake. Despite both steroids enhancing uptake, degradation was impeded, particularly with Gcs. Distinct differences between the two steroids were observed in their effect on toxin production and cell viability. Gc dose-dependently increased toxicity and potentiated Aβ induction of nitric oxide, while E2 promoted cell viability and inhibited Aβ induction of nitric oxide. The steroid enhancement of μglial uptake and impedence of degradation observed in vitro were consistent with observations from in vivo studies. In the brains of Aβ-infused rats, the μglial staining in entorhinal cortex layer 3, not associated with Aβ deposits was increased in response to Aβ infusion and this effect was blocked by feeding rats prednisolone. In contrast, E2 enhanced μglial staining in Aβ-infused rats. Aβ-immunoreactive (ir) deposits were quantitatively smaller, appeared denser, and were associated with robust μglial responses. Despite the fact that steroid produced a smaller more focal deposit, total extracted Aβ in cortical homogenate was elevated. Together, the in vivo and in vitro data support a role for steroids in plaque compaction. Our data are also consistent with the hypothesis that although E2 is less potent than Gc in impeding Aβ degradation, long term exposure to both steroids could reduce Aβ clearance and clinical utility. These data showing Gc potentiation of Aβ-induced μglial toxins may help explain the lack of epidemiological correlation for AD. The failure of both steroids to accelerate Aβ degradation may explain their lack of efficacy for treatment of AD.     

Mechanisms of hybrid oligomer formation in the pathogenesis of combined Alzheimer's and Parkinson's diseases

Background

Misfolding and pathological aggregation of neuronal proteins has been proposed to play a critical role in the pathogenesis of neurodegenerative disorders. Alzheimer''s disease (AD) and Parkinson''s disease (PD) are frequent neurodegenerative diseases of the aging population. While progressive accumulation of amyloid β protein (Aβ) oligomers has been identified as one of the central toxic events in AD, accumulation of α-synuclein (α-syn) resulting in the formation of oligomers and protofibrils has been linked to PD and Lewy body Disease (LBD). We have recently shown that Aβ promotes α-syn aggregation and toxic conversion in vivo, suggesting that abnormal interactions between misfolded proteins might contribute to disease pathogenesis. However the molecular characteristics and consequences of these interactions are not completely clear.

Methodology/Principal Findings

In order to understand the molecular mechanisms involved in potential Aβ/α-syn interactions, immunoblot, molecular modeling, and in vitro studies with α-syn and Aβ were performed. We showed in vivo in the brains of patients with AD/PD and in transgenic mice, Aβ and α-synuclein co-immunoprecipitate and form complexes. Molecular modeling and simulations showed that Aβ binds α-syn monomers, homodimers, and trimers, forming hybrid ring-like pentamers. Interactions occurred between the N-terminus of Aβ and the N-terminus and C-terminus of α-syn. Interacting α-syn and Aβ dimers that dock on the membrane incorporated additional α-syn molecules, leading to the formation of more stable pentamers and hexamers that adopt a ring-like structure. Consistent with the simulations, under in vitro cell-free conditions, Aβ interacted with α-syn, forming hybrid pore-like oligomers. Moreover, cells expressing α-syn and treated with Aβ displayed increased current amplitudes and calcium influx consistent with the formation of cation channels.

Conclusion/Significance

These results support the contention that Aβ directly interacts with α-syn and stabilized the formation of hybrid nanopores that alter neuronal activity and might contribute to the mechanisms of neurodegeneration in AD and PD. The broader implications of such hybrid interactions might be important to the pathogenesis of other disorders of protein misfolding.     

Protective effect of the xanthate, D609, on Alzheimer's amyloid beta-peptide (1-42)-induced oxidative stress in primary neuronal cells

Tricyclodecan-9-yl-xanthogenate (D609) is an inhibitor of phosphatidylcholine-specific phospholipase C, and this agent also has been reported to protect rodents against oxidative damage induced by ionizing radiation. Previously, we showed that D609 mimics glutathione (GSH) functions and that a disulfide is formed upon oxidation of D609 and the resulting dixanthate is a substrate for GSH reductase, regenerating D609. Considerable attention has been focused on increasing the intracellular GSH levels in many diseases, including Alzheimer's disease (AD). Amyloid β-peptide [Aβ(1-42)], elevated in AD brain, is associated with oxidative stress and toxicity. The present study aimed to investigate the protective effects of D609 on Aβ(1-42)-induced oxidative cell toxicity in cultured neurons. Decreased cell survival in neuronal cultures treated with Aβ(1-42) correlated with increased free radical production measured by dichlorofluorescein fluorescence and an increase in protein oxidation (protein carbonyl, 3-nitrotyrosine) and lipid peroxidation (4-hydroxy-2-nonenal) formation. Pretreatment of primary hippocampal cultures with D609 significantly attenuated Aβ(1-42)-induced cytotoxicity, intracellular ROS accumulation, protein oxidation, lipid peroxidation and apoptosis. Methylated D609, with the thiol functionality no longer able to form the disulfide upon oxidation, did not protect neuronal cells against Aβ(1-42)-induced oxidative stress. Our results suggest that D609 exerts protective effects against Aβ(1-42) toxicity by modulating oxidative stress. These results may be of importance for the treatment of AD and other oxidative stress-related diseases.     

The Neuroendocrine Protein 7B2 Suppresses the Aggregation of Neurodegenerative Disease-related Proteins

Neurodegenerative diseases such as Alzheimer (AD) and Parkinson (PD) are characterized by abnormal aggregation of misfolded β-sheet-rich proteins, including amyloid-β (Aβ)-derived peptides and tau in AD and α-synuclein in PD. Correct folding and assembly of these proteins are controlled by ubiquitously expressed molecular chaperones; however, our understanding of neuron-specific chaperones and their involvement in the pathogenesis of neurodegenerative diseases is limited. We here describe novel chaperone-like functions for the secretory protein 7B2, which is widely expressed in neuronal and endocrine tissues. In in vitro experiments, 7B2 efficiently prevented fibrillation and formation of Aβ_1–42, Aβ_1–40, and α-synuclein aggregates at a molar ratio of 1:10. In cell culture experiments, inclusion of recombinant 7B2, either in the medium of Neuro-2A cells or intracellularly via adenoviral 7B2 overexpression, blocked the neurocytotoxic effect of Aβ_1–42 and significantly increased cell viability. Conversely, knockdown of 7B2 by RNAi increased Aβ_1–42-induced cytotoxicity. In the brains of APP/PSEN1 mice, a model of AD amyloidosis, immunoreactive 7B2 co-localized with aggregation-prone proteins and their respective aggregates. Furthermore, in the hippocampus and substantia nigra of human AD- and PD-affected brains, 7B2 was highly co-localized with Aβ plaques and α-synuclein deposits, strongly suggesting physiological association. Our data provide insight into novel functions of 7B2 and establish this neural protein as an anti-aggregation chaperone associated with neurodegenerative disease.     

Prion Protein-mediated Toxicity of Amyloid-β Oligomers Requires Lipid Rafts and the Transmembrane LRP1

Soluble oligomers of the amyloid-β (Aβ) peptide cause neurotoxicity, synaptic dysfunction, and memory impairments that underlie Alzheimer disease (AD). The cellular prion protein (PrP^C) was recently identified as a high affinity neuronal receptor for Aβ oligomers. We report that fibrillar Aβ oligomers recognized by the OC antibody, which have been shown to correlate with the onset and severity of AD, bind preferentially to cells and neurons expressing PrP^C. The binding of Aβ oligomers to cell surface PrP^C, as well as their downstream activation of Fyn kinase, was dependent on the integrity of cholesterol-rich lipid rafts. In SH-SY5Y cells, fluorescence microscopy and co-localization with subcellular markers revealed that the Aβ oligomers co-internalized with PrP^C, accumulated in endosomes, and subsequently trafficked to lysosomes. The cell surface binding, internalization, and downstream toxicity of Aβ oligomers was dependent on the transmembrane low density lipoprotein receptor-related protein-1 (LRP1). The binding of Aβ oligomers to cell surface PrP^C impaired its ability to inhibit the activity of the β-secretase BACE1, which cleaves the amyloid precursor protein to produce Aβ. The green tea polyphenol (−)-epigallocatechin gallate and the red wine extract resveratrol both remodeled the fibrillar conformation of Aβ oligomers. The resulting nonfibrillar oligomers displayed significantly reduced binding to PrP^C-expressing cells and were no longer cytotoxic. These data indicate that soluble, fibrillar Aβ oligomers bind to PrP^C in a conformation-dependent manner and require the integrity of lipid rafts and the transmembrane LRP1 for their cytotoxicity, thus revealing potential targets to alleviate the neurotoxic properties of Aβ oligomers in AD.     

Modulation of γ-secretase activity by multiple enzyme-substrate interactions: implications in pathogenesis of Alzheimer's disease

Background

We describe molecular processes that can facilitate pathogenesis of Alzheimer''s disease (AD) by analyzing the catalytic cycle of a membrane-imbedded protease γ-secretase, from the initial interaction with its C99 substrate to the final release of toxic Aβ peptides.

Results

The C-terminal AICD fragment is cleaved first in a pre-steady-state burst. The lowest Aβ42/Aβ40 ratio is observed in pre-steady-state when Aβ40 is the dominant product. Aβ42 is produced after Aβ40, and therefore Aβ42 is not a precursor for Aβ40. The longer more hydrophobic Aβ products gradually accumulate with multiple catalytic turnovers as a result of interrupted catalytic cycles. Saturation of γ-secretase with its C99 substrate leads to 30% decrease in Aβ40 with concomitant increase in the longer Aβ products and Aβ42/Aβ40 ratio. To different degree the same changes in Aβ products can be observed with two mutations that lead to an early onset of AD, ΔE9 and G384A. Four different lines of evidence show that γ-secretase can bind and cleave multiple substrate molecules in one catalytic turnover. Consequently depending on its concentration, NotchΔE substrate can activate or inhibit γ-secretase activity on C99 substrate. Multiple C99 molecules bound to γ-secretase can affect processive cleavages of the nascent Aβ catalytic intermediates and facilitate their premature release as the toxic membrane-imbedded Aβ-bundles.

Conclusions

Gradual saturation of γ-secretase with its substrate can be the pathogenic process in different alleged causes of AD. Thus, competitive inhibitors of γ-secretase offer the best chance for a successful therapy, while the noncompetitive inhibitors could even facilitate development of the disease by inducing enzyme saturation at otherwise sub-saturating substrate. Membrane-imbedded Aβ-bundles generated by γ-secretase could be neurotoxic and thus crucial for our understanding of the amyloid hypothesis and AD pathogenesis.     

Amyloid-β Acts as a Regulator of Neurotransmitter Release Disrupting the Interaction between Synaptophysin and VAMP2

Background

It is becoming increasingly evident that deficits in the cortex and hippocampus at early stages of dementia in Alzheimer’s disease (AD) are associated with synaptic damage caused by oligomers of the toxic amyloid-β peptide (Aβ42). However, the underlying molecular and cellular mechanisms behind these deficits are not fully understood. Here we provide evidence of a mechanism by which Aβ42 affects synaptic transmission regulating neurotransmitter release.

Methodology/Findings

We first showed that application of 50 nM Aβ42 in cultured neurones is followed by its internalisation and translocation to synaptic contacts. Interestingly, our results demonstrate that with time, Aβ42 can be detected at the presynaptic terminals where it interacts with Synaptophysin. Furthermore, data from dissociated hippocampal neurons as well as biochemical data provide evidence that Aβ42 disrupts the complex formed between Synaptophysin and VAMP2 increasing the amount of primed vesicles and exocytosis. Finally, electrophysiology recordings in brain slices confirmed that Aβ42 affects baseline transmission.

Conclusions/Significance

Our observations provide a necessary and timely insight into cellular mechanisms that underlie the initial pathological events that lead to synaptic dysfunction in Alzheimer’s disease. Our results demonstrate a new mechanism by which Aβ42 affects synaptic activity.     

Eight-residue Abeta peptides inhibit the aggregation and enzymatic activity of Abeta42

Insoluble Aβ1–42 is the main component of the amyloid plaque. We have previously demonstrated that exposure to low pH can confer the molten globule state on soluble Aβ1–42 in vitro [Biochem. J. 361 (2000) 547] and unfolding experiments with guadinine hydrochloride (GdnHCl) have now confirmed this observation. The molten globule state of the protein has many biological properties and understanding the mechanisms of its formation is an important step in devising a therapeutic strategy for Alzheimer's disease (AD). We therefore investigated the ability of a series of synthetic eight-residue peptides derived from Aβ1–42 to inhibit the acid-induced aggregation of Aβ1–42 and identified the potent peptides to be Aβ15–22, Aβ16–23 and Aβ17–24. A1-antichymotrypsin, a member of the serine proteinase inhibitor (serpin) family is another major component of the amyloid plaque. In the present study, we investigated the proteolytic activity of Aβ1–42 against casein at different pHs. Chemical modification of amino acid residues in Aβ1–42 indicated that serine and histidine residues, but not aspartic acid, are necessary for enzymatic activity, suggesting that it is a serine proteinase. Amino acid substitution studies indicate that glutamic acids at positions 11 and 22 participate indirectly in proteolysis and we surmise that amino acid residues 29–42 are required to stabilize the conformer. A study of metal ions suggested that Cu²⁺ affected the enzymatic activity, but Zn²⁺ and Fe²⁺ did not. Interestingly, Aβ14–21 and Aβ15–22 were the only peptides that inhibited the proteolytic activity of Aβ42. Therefore, Aβ15–22 may control both aggregation of Aβ1–42 at acidic pH and its proteolytic activity at neutral pH. Consequently, we suggest that it may be of use in the therapy of Alzheimer's disease.