Aβ Oligomers and Fibrillar Aggregates Induce Different Apoptotic Pathways in LAN5 Neuroblastoma Cell Cultures

Fibril deposit formation of amyloid β-protein (Aβ) in the brain is a hallmark of Alzheimer's disease (AD). Increasing evidence suggests that toxicity is linked to diffusible Aβ oligomers, which have been found in soluble brain extracts of AD patients, rather than to insoluble fibers. Here we report a study of the toxicity of two distinct forms of recombinant Aβ small oligomers and fibrillar aggregates to simulate the action of diffusible Aβ oligomers and amyloid plaques on neuronal cells. Different techniques, including dynamic light scattering, fluorescence, and scanning electron microscopy, have been used to characterize the two forms of Aβ. Under similar conditions and comparable incubation times in neuroblastoma LAN5 cell cultures, oligomeric species obtained from Aβ peptide are more toxic than fibrillar aggregates. Both oligomers and aggregates are able to induce neurodegeneration by apoptosis activation, as demonstrated by TUNEL assay and Hoechst staining assays. Moreover, we show that aggregates induce apoptosis by caspase 8 activation (extrinsic pathway), whereas oligomers induce apoptosis principally by caspase 9 activation (intrinsic pathway). These results are confirmed by cytochrome c release, almost exclusively detected in the cytosolic fraction of LAN5 cells treated with oligomers. These findings indicate an active and direct interaction between oligomers and the cellular membrane, and are consistent with internalization of the oligomeric species into the cytosol.     

Association between frontal cortex oxidative damage and beta-amyloid as a function of age in Down syndrome

Down syndrome (DS) is the most common genetic cause of intellectual disability in children, and the number of adults with DS reaching old age is increasing. By the age of 40 years, virtually all people with DS have sufficient neuropathology for a postmortem diagnosis of Alzheimer disease (AD). Trisomy 21 in DS leads to an overexpression of many proteins, of which at least two are involved in oxidative stress and AD: superoxide dismutase 1 (SOD1) and amyloid precursor protein (APP). In this study, we tested the hypothesis that DS brains with neuropathological hallmarks of AD have more oxidative and nitrosative stress than those with DS but without significant AD pathology, as compared with similarly aged-matched non-DS controls. The frontal cortex was examined in 70 autopsy cases (n = 29 control and n = 41 DS). By ELISA, we quantified soluble and insoluble Aβ40 and Aβ42, as well as oligomers. Oxidative and nitrosative stress levels (protein carbonyls, 4-hydroxy-2-trans-nonenal (HNE)-bound proteins, and 3-nitrotyrosine) were measured by slot-blot. We found that soluble and insoluble amyloid beta peptide (Aβ) and oligomers increase as a function of age in DS frontal cortex. Of the oxidative stress markers, HNE-bound proteins were increased overall in DS. Protein carbonyls were correlated with Aβ40 levels. These results suggest that oxidative damage, but not nitrosative stress, may contribute to the onset and progression of AD pathogenesis in DS. Conceivably, treatment with antioxidants may provide a point of intervention to slow pathological alterations in DS.     

Protective effects of EphB2 on Aβ1–42 oligomer-induced neurotoxicity and synaptic NMDA receptor signaling in hippocampal neurons

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized pathologically by the abnormal deposition of extracellular amyloid-β (Aβ) oligomers. However, the nature and precise mechanism of the toxicity of Aβ oligomers are not clearly understood. Aβ oligomers have been previously shown to cause a major loss of EphB2, a member of the EphB family of receptor tyrosine kinases. To determine the effect of EphB2 on Aβ oligomer-induced neurotoxicity and the underlying molecular mechanisms, we examined the EphB2 gene in cultured hippocampal neurons. Using a cellular model of AD, Aβ_1–42 oligomers were confirmed to induce neurotoxicity in a time-dependent manner and result in a major decrease of EphB2. EphB2 overexpression could prevent the neurotoxicity of hippocampal neurons from exposure to Aβ_1–42 oligomers for 1 h. Further analysis revealed that EphB2 overexpression increased synaptic NR1 and NR2B expression in Aβ_1–42 oligomer-treated neurons. Moreover, EphB2 overexpression prevented Aβ_1–42 oligomer-induced downregulation of dephosphorylated p38 MAPK and phosphorylated CREB. Together, these results suggest that EphB2 is a factor which protects hippocampal neurons against the toxicity of Aβ_1–42 oligomers, and we infer that the protection of EphB2 is achieved by increasing the synaptic NMDA receptor level and downstream p38 MAPK and CREB signaling in hippocampal neurons. This study provides new molecular insights into the neuroprotective effect of EphB2 and highlights its potential therapeutic role in the management of AD.     

Anthocyanin-enriched bilberry and blackcurrant extracts modulate amyloid precursor protein processing and alleviate behavioral abnormalities in the APP/PS1 mouse model of Alzheimer's disease

A growing body of epidemiological evidence suggests that fruit and vegetable juices containing various phenolic compounds can reduce the risk of Alzheimer's disease (AD). As the altered amyloid precursor protein (APP) processing leading to increased β-amyloid (Aβ) production is a key pathogenic feature of AD, we elucidated the effects of different polyphenols on neuroprotection and APP processing under different in vitro stress conditions. The effects of these compounds were also investigated in transgenic AD mice (APdE9). Free radical toxicity and apoptosis were induced in human SH-SY5Y neuroblastoma cells overexpressing APP751. Menadione-induced production of reactive oxygen species was significantly decreased upon treatment with myricetin, quercetin or anthocyanin-rich extracts in a dose-dependent manner. However, these extracts did not affect caspase-3 activation, APP processing or Aβ levels upon staurosporine-induced apoptosis. APdE9 mice fed with anthocyanin-rich bilberry or blackcurrant extracts showed decreased APP C-terminal fragment levels in the cerebral cortex as compared to APdE9 mice on the control diet. Soluble Aβ40 and Aβ42 levels were significantly decreased in bilberry-fed mice as compared to blackcurrant-fed mice. Conversely, the ratio of insoluble Aβ42/40 was significantly decreased in blackcurrant-fed mice relative to bilberry-fed mice. Both berry diets alleviated the spatial working memory deficit of aged APdE9 mice as compared to mice on the control diet. There were no changes in the expression or phosphorylation status of tau in APdE9 mice with respect to diet. These data suggest that anthocyanin-rich bilberry and blackcurrant diets favorably modulate APP processing and alleviate behavioral abnormalities in a mouse model of AD.     

How Fluorescent Tags Modify Oligomer Size Distributions of the Alzheimer Peptide

Within the complex aggregation process of amyloidogenic peptides into fibrils, early stages of aggregation play a central role and reveal fundamental properties of the underlying mechanism of aggregation. In particular, low-molecular-weight aggregates of the Alzheimer amyloid-β peptide (Aβ) have attracted increasing interest because of their role in cytotoxicity and neuronal apoptosis, typical of aggregation-related diseases. One of the main techniques used to characterize oligomeric stages is fluorescence spectroscopy. To this end, Aβ peptide chains are functionalized with fluorescent tags, often covalently bound to the disordered N-terminus region of the peptide, with the assumption that functionalization and presence of the fluorophore will not modify the process of self-assembly nor the final fibrillar structure. In this investigation, we systematically study the effects of four of the most commonly used fluorophores on the aggregation of Aβ (1–40). Time-resolved and single-molecule fluorescence spectroscopy have been chosen to monitor the oligomer populations at different fibrillation times, and transmission electron microscopy, atomic force microscopy and x-ray diffraction to investigate the structure of mature fibrils. Although the structures of the fibrils were only slightly affected by the fluorescent tags, the sizes of the detected oligomeric species varied significantly depending on the chosen fluorophore. In particular, we relate the presence of high-molecular-weight oligomers of Aβ (1–40) (as found for the fluorophores HiLyte 647 and Atto 655) to net-attractive, hydrophobic fluorophore-peptide interactions, which are weak in the case of HiLyte 488 and Atto 488. The latter leads for Aβ (1–40) to low-molecular-weight oligomers only, which is in contrast to Aβ (1–42). The disease-relevant peptide Aβ (1–42) displays high-molecular-weight oligomers even in the absence of significant attractive fluorophore-peptide interactions. Hence, our findings reveal the potentially high impact of the properties of fluorophores on transient aggregates, which needs to be included in the interpretation of experimental data of oligomers of fluorescently labeled peptides.     

Effects of NMDAR Antagonist on the Regulation of P-MARCKS Protein to Aβ<Subscript>1−42</Subscript> Oligomers Induced Neurotoxicity

Alzheimer’s disease (AD) is a well-known neurodegenerative disease. Deposition of β-amyloid protein (Aβ) oligomers plays a crucial role in the disease progression. Previous studies showed that toxicity induced by Aβ oligomers in cultured neurons and adult rat brain was partially mediated by activation of glutamatergic N-methyl-d-aspartate receptors (NMDAR). Additionally, memantine, a noncompetitive NMDAR antagonist, can significantly improve cognitive functions in some AD patients. However, little is currently known about the potential role of NMDAR antagonist on the regulation of P-MARCKS protein to Aβ_1?42 oligomers induced neurotoxicity. The protective effect and mechanism of NMDAR antagonist on primary neurons exposed to Aβ_1?42 oligomers were investigated in the study. We have defined that the Aβ_1?42 treatment decreased cell viability and increased apoptosis. Moreover, Aβ_1?42 oligomers exposure increased P-MARCKS and PIP2 expressions, while decreased SYP expression. However, NMDAR antagonist pretreatment ameliorates Aβ_1?42 oligomers induced neuronal apoptosis and partially reverses the expression of P-MARCKS, PIP2 and SYP. In conclusion, NMDAR antagonist may ameliorate neurotoxicity induced by Aβ_1?42 oligomers through reducing neuronal apoptosis and protecting synaptic plasticity in rat primary neurons. The mechanism involved may be mediated by the variation of protein P-MARCKS.     

High-molecular weight Aβ oligomers and protofibrils are the predominant Aβ species in the native soluble protein fraction of the AD brain

Alzheimer's disease (AD) is characterized by the aggregation and deposition of amyloid β protein (Aβ) in the brain. Soluble Aβ oligomers are thought to be toxic. To investigate the predominant species of Aβ protein that may play a role in AD pathogenesis, we performed biochemical analysis of AD and control brains. Sucrose buffer-soluble brain lysates were characterized in native form using blue native (BN)-PAGE and also in denatured form using SDS-PAGE followed by Western blot analysis. BN-PAGE analysis revealed a high-molecular weight smear (>1000 kD) of Aβ(42) -positive material in the AD brain, whereas low-molecular weight and monomeric Aβ species were not detected. SDS-PAGE analysis, on the other hand, allowed the detection of prominent Aβ monomer and dimer bands in AD cases but not in controls. Immunoelectron microscopy of immunoprecipitated oligomers and protofibrils/fibrils showed spherical and protofibrillar Aβ-positive material, thereby confirming the presence of high-molecular weight Aβ (hiMWAβ) aggregates in the AD brain. In vitro analysis of synthetic Aβ(40) - and Aβ(42) preparations revealed Aβ fibrils, protofibrils, and hiMWAβ oligomers that were detectable at the electron microscopic level and after BN-PAGE. Further, BN-PAGE analysis exhibited a monomer band and less prominent low-molecular weight Aβ (loMWAβ) oligomers. In contrast, SDS-PAGE showed large amounts of loMWAβ but no hiMWAβ(40) and strikingly reduced levels of hiMWAβ(42) . These results indicate that hiMWAβ aggregates, particularly Aβ(42) species, are most prevalent in the soluble fraction of the AD brain. Thus, soluble hiMWAβ aggregates may play an important role in the pathogenesis of AD either independently or as a reservoir for release of loMWAβ oligomers.     

Size-dependent neurotoxicity of β-amyloid oligomers

The link between the size of soluble amyloid β (Aβ) oligomers and their toxicity to rat cerebellar granule cells (CGC) was investigated. Variation in conditions during in vitro oligomerization of Aβ_1-42 resulted in peptide assemblies with different particle size as measured by atomic force microscopy and confirmed by dynamic light scattering and fluorescence correlation spectroscopy. Small oligomers of Aβ_1-42 with a mean particle z-height of 1-2 nm exhibited propensity to bind to phospholipid vesicles and they were the most toxic species that induced rapid neuronal necrosis at submicromolar concentrations whereas the bigger aggregates (z-height above 4-5 nm) did not bind vesicles and did not cause detectable neuronal death. A similar neurotoxic pattern was also observed in primary cultures of cortex neurons whereas Aβ₁₋₄₂ oligomers, monomers and fibrils were non-toxic to glial cells in CGC cultures or macrophage J774 cells. However, both oligomeric forms of Aβ_1-42 induced reduction of neuronal cell densities in the CGC cultures.     

Intracellular amyloid-beta 1-42, but not extracellular soluble amyloid-beta peptides, induces neuronal apoptosis

Alzheimer disease (AD), the most frequent cause of dementia, is characterized by an important neuronal loss. A typical histological hallmark of AD is the extracellular deposition of beta-amyloid peptide (A beta), which is produced by the cleavage of the amyloid precursor protein (APP). Most of the gene mutations that segregate with the inherited forms of AD result in increasing the ratio of A beta 42/A beta 40 production. A beta 42 also accumulates in neurons of AD patients. Altogether, these data strongly suggest that the neuronal production of A beta 42 is a critical event in AD, but the intraneuronal A beta 42 toxicity has never been demonstrated. Here, we report that the long term expression of human APP in rat cortical neurons induces apoptosis. Although APP processing leads to production of extracellular A beta 1-40 and soluble APP, these extracellular derivatives do not induce neuronal death. On the contrary, neurons undergo apoptosis as soon as they accumulate intracellular A beta 1-42 following the expression of full-length APP or a C-terminal deleted APP isoform. The inhibition of intraneuronal A beta 1-42 production by a functional gamma-secretase inhibitor increases neuronal survival. Therefore, the accumulation of intraneuronal A beta 1-42 is the key event in the neurodegenerative process that we observed.     

Interaction of dipeptydil peptidase IV with amyloid peptides

The aggregates of amyloid beta peptides (Aβs) are regarded as one of the main pathological hallmarks of Alzheimer’s disease (AD). An imbalance between the rates of synthesis and clearance of Aβs is considered to be a possible cause for the onset of AD. Dipeptidyl peptidases II and IV (DPPII and DPPIV) are serine proteases removing N-terminal dipeptides from polypeptides and proteins with proline or alanine on the penultimate position. Alanine is an N-terminal penultimate residue in Аβs, and we presumed that DPPII and DPPIV could cleave them. The results of present in vitro research demonstrate for the first time the ability of DPPIV to truncate the commercial Aβ40 and Aβ42 peptides, to hinder the fibril formation by them and to participate in the disaggregation of preformed fibrils of these peptides. The increase of absorbance at 334 nm due to complex formation between primary amines with o-phtalaldehyde was used to show cleaving of Aβ40 and Aβ42. The time-dependent increase of the quantity of primary amines during incubation of peptides in the presence of DPPIV suggested their truncation by DPPIV, but not by DPPII. The parameters of the enzymatic breakdown by DPPIV were determined for Aβ40 (K_m = 37.5 μM, k_cat/K_m = 1.7 × 10³ M⁻¹sec⁻¹) and Aβ42 (K_m = 138.4 μM, k_cat/K_m = 1.90 × 10² M⁻¹sec⁻¹). The aggregation-disaggregation of peptides was controlled by visualization on transmission electron microscope and by Thioflavin-T fluorescence on spectrofluorimeter and fluorescent microscope. DPPIV hindered the peptide aggregation/fibrillation during 3-4 days incubation in 20 mM phosphate buffer, pH 7.4, 37 °C by 50–80%. Ovalbumin, BSA and DPPII did not show this effect. In the presence of DPPIV, the preformed fibrils were disaggregated by 30–40%. Conclusion: for the first time it was shown that the Aβ40 and Aβ42 are substrates of DPPIV. DPPIV prohibits the fibrillation of peptides and promotes disaggregation of their preformed aggregates.     

N-Terminal pyroglutamate formation of Aβ38 and Aβ40 enforces oligomer formation and potency to disrupt hippocampal long-term potentiation

Pyroglutamate (pGlu)-modified amyloid peptides have been identified in sporadic and familial forms of Alzheimer's disease (AD) and the inherited disorders familial British and Danish Dementia (FBD and FDD). In this study, we characterized the aggregation of amyloid-β protein Aβ37, Aβ38, Aβ40, Aβ42 and ADan species in vitro, which were modified by N-terminal pGlu (pGlu-Aβ3-x, pGlu-ADan) or possess the intact N-terminus (Aβ1-x, ADan). The pGlu-modification confers rapid formation of oligomers and short fibrillar aggregates. In accordance with these observations, the pGlu-modified Aβ38, Αβ40 and Αβ42 species inhibit hippocampal long term potentiation of synaptic response, but pGlu-Aβ3-42 showing the highest effect. Among the unmodified Aβ peptides, only Aβ1-42 exhibites such propensity, which was similar to pGlu-Aβ3-38 and pGlu-Aβ3-40. Likewise, the amyloidogenic peptide pGlu-ADan impaired synaptic potentiation more pronounced than N-terminal unmodified ADan. The results were validated using conditioned media from cultivated HEK293 cells, which express APP variants favoring the formation of Aβ1-x, Aβ3-x or N-truncated pGlu-Aβ3-x species. Hence, we show that the ability of different amyloid peptides to impair synaptic function apparently correlates to their potential to form oligomers as a common mechanism. The pGlu-modification is apparently mediating a higher surface hydrophobicity, as shown by 1-anilinonaphtalene-8-sulfonate fluorescence, which enforces potential to interfere with neuronal physiology.     

Menadione sodium bisulfite inhibits the toxic aggregation of amyloid-β(1–42)

Protein misfolding and aggregation are associated with amyloidosis. The toxic aggregation of amyloid-β 1–42 (Aβ42) may disrupt cell membranes and lead to cell death and is thus regarded as a contributing factor in Alzheimer's disease (AD). 1,4-naphthoquinone (NQ) has been shown to exhibit strong anti-aggregation effects on amyloidogenic proteins such as insulin and α-synuclein; however, its high toxicity and poor solubility limit its clinical application. Menadione sodium bisulfite (MSB, also known as vitamin K3), is used clinically in China to treat hemorrhagic diseases caused by vitamin K deficiency and globally as a vitamin K supplement. We hypothesized that MSB could inhibit amyloid formation since its backbone structure is similar to NQ. To test our hypothesis, we first investigated the effects of MSB on Aβ42 amyloid formation in vitro. We found that MSB inhibited Aβ42 amyloid formation in a dose dependent manner, delayed the secondary structural conversion of Aβ42 from random coil to ordered β-sheet, and attenuated the ability of Aβ42 aggregates to disrupt membranes; moreover, the quinone backbone rather than lipophilicity is esstial for the inhibitory effects of MSB. Next, in cells expressing a pathogenic APP mutation (Osaka mutation) that results in the formation of intraneuronal Aβ oligomers, MSB inhibited the intracellular aggregation of Aβ. Moreover, MSB treatment significantly extended the life span of Caenorhabditis elegans CL2120, a strain that expresses human Aβ42. Together, these results suggest that MSB and its derivatives may be further explored as potential therapeutic agents for the prevention or treatment of AD.     

Carbon nanotube prevents the secondary structure formation of amyloid-β trimers: an all-atom molecular dynamics study

Abstract

Increasing evidence shows that the formation of misfolded aggregates amyloid-β (Aβ) peptide is associated with the Alzheimer’s disease (AD). Recent experiments reveal a significant correlation of the amount of trimer species bound to neurons with increasing neuro-toxicity. Our previous computational study demonstrated that carbon nanotubes (CNT) can inhibit effectively the formation of β-sheet-rich oligomers of Aβ(16-22) – a hydrophobic peptide essential for Aβ fibrillization. However, the influence of CNT on the oligomers formed by full-length Aβ remains elusive. In this study, we have investigated the conformational dynamics of Aβ(1-42) trimer, built from an NMR structure of α-helical monomer, in the absence and presence of a single-walled carbon nanotube (SWCNT). Our simulations demonstrate that SWCNT can significantly hinder the trimerisation and prevents the secondary structure formation of Aβ(1-42) peptide. Hydrophobic and aromatic stacking interactions between SWCNT and Aβ play important roles in the secondary structure formation of the Aβ trimer. This study reveals a complete picture of the detailed preventable mechanism of full-length Aβ(1-42) by SWCNT, providing theoretical insights into the development of drug candidates of AD.     

Isolation of synaptic terminals from Alzheimer's disease cortex

Amyloid beta (Aβ) oligomers and phosphorylated tau (p-tau) aggregates are increasingly identified as potential toxic intermediates in Alzheimer's disease (AD). In cortical AD synapses, p-tau co-localizes with Aβ, but the Aβ and p-tau peptide species responsible for synaptic dysfunction and demise remains unclear. The present experiments were designed to use high-speed cell sorting techniques to purify synaptosome population based on size, and then extend the method to physically isolate Aβ-positive synaptosomes with the goal of understanding the nature of Aβ and tau pathology in AD synapses. To examine the purity of size-gated synaptosomes, samples were first gated on size; particles with sizes between 0.5 and 1.5 microns were collected. Electron microscopy documented a homogenous population of spherical particles with internal vesicles and synaptic densities. Next, size-gated synaptosomes positive for Aβ were collected by fluorescence activated sorting and then analyzed by immunoblotting techniques. Sorted Aβ-positive synaptosomes were enriched for amyloid precursor protein (APP) and for Aβ oligomers and aggregates; immunolabeling for p-tau showed a striking accumulation of p-tau aggregates compared to the original homogenate and purified synaptosomes. These results confirm co-localization of Aβ and p-tau within individual synaptic terminals and provide proof of concept for the utility of flow sorting synaptosomes.     

Amyloid β binds procaspase-9 to inhibit assembly of Apaf-1 apoptosome and intrinsic apoptosis pathway

Apoptosis is essential in the death process induced by Amyloid-β (Aβ), a major constituent of diffuse plaques found in Alzheimer's disease patients. However, we have found that caspase activation and cell death induced by staurosporine, employed to induce the intrinsic mitochondria-dependent apoptotic pathway, were significantly reduced by 42 amino-acid Aβ42, implying that the peptide also has a negative effect on the apoptotic process. The inhibitory effect of Aβ42 on the apoptotic pathway is associated with its interaction with procaspase-9 and consequent inhibition of Apaf-1 apoptosome assembly. We detected the inhibitory effect in the early stage (< 8 h) of apoptosis, but later caspase activation becomes obvious. Thus we inferred that the inhibitory process on apoptosis begins at an early stage, and the later robust activation surpasses it. We propose that the apoptotic manifestation in Aβ-treated cells is a combined consequence of those anti- and pro-apoptotic processes.     

Docosahexaenoic acid-derived neuroprotectin D1 induces neuronal survival via secretase- and PPARγ-mediated mechanisms in Alzheimer's disease models

Neuroprotectin D1 (NPD1) is a stereoselective mediator derived from the omega-3 essential fatty acid docosahexaenoic acid (DHA) with potent inflammatory resolving and neuroprotective bioactivity. NPD1 reduces Aβ42 peptide release from aging human brain cells and is severely depleted in Alzheimer's disease (AD) brain. Here we further characterize the mechanism of NPD1's neurogenic actions using 3xTg-AD mouse models and human neuronal-glial (HNG) cells in primary culture, either challenged with Aβ42 oligomeric peptide, or transfected with beta amyloid precursor protein (βAPP)(sw) (Swedish double mutation APP695(sw), K595N-M596L). We also show that NPD1 downregulates Aβ42-triggered expression of the pro-inflammatory enzyme cyclooxygenase-2 (COX-2) and of B-94 (a TNF-α-inducible pro-inflammatory element) and apoptosis in HNG cells. Moreover, NPD1 suppresses Aβ42 peptide shedding by down-regulating β-secretase-1 (BACE1) while activating the α-secretase ADAM10 and up-regulating sAPPα, thus shifting the cleavage of βAPP holoenzyme from an amyloidogenic into the non-amyloidogenic pathway. Use of the thiazolidinedione peroxisome proliferator-activated receptor gamma (PPARγ) agonist rosiglitazone, the irreversible PPARγ antagonist GW9662, and overexpressing PPARγ suggests that the NPD1-mediated down-regulation of BACE1 and Aβ42 peptide release is PPARγ-dependent. In conclusion, NPD1 bioactivity potently down regulates inflammatory signaling, amyloidogenic APP cleavage and apoptosis, underscoring the potential of this lipid mediator to rescue human brain cells in early stages of neurodegenerations.     

A novel peptide derived from vascular endothelial growth factor prevents amyloid beta aggregation and toxicity

Amyloid-β oligomers (Aβo) are the most pathologically relevant Aβ species in Alzheimer's disease (AD), because they induce early synaptic dysfunction that leads to learning and memory impairments. In contrast, increasing VEGF (Vascular Endothelial Growth Factor) brain levels have been shown to improve learning and memory processes, and to alleviate Aβ-mediated synapse dysfunction. Here, we designed a new peptide, the blocking peptide (BP), which is derived from an Aβo-targeted domain of the VEGF protein, and investigated its effect on Aβ-associated toxicity. Using a combination of biochemical, 3D and ultrastructural imaging, and electrophysiological approaches, we demonstrated that BP strongly interacts with Aβo and blocks Aβ fibrillar aggregation process, leading to the formation of Aβ amorphous aggregates. BP further impedes the formation of structured Aβo and prevents their pathogenic binding to synapses. Importantly, acute BP treatment successfully rescues long-term potentiation (LTP) in the APP/PS1 mouse model of AD, at an age when LTP is highly impaired in hippocampal slices. Moreover, BP is also able to block the interaction between Aβo and VEGF, which suggests a dual mechanism aimed at both trapping Aβo and releasing VEGF to alleviate Aβo-induced synaptic damage. Our findings provide evidence for a neutralizing effect of the BP on Aβ aggregation process and pathogenic action, highlighting a potential new therapeutic strategy.     

Scavenging amyloid oligomers from neurons with silica nanobowls: Implications for amyloid diseases

Amyloid-β (Aβ) oligomers are toxic species implicated in Alzheimer’s disease (AD). The prevailing hypothesis implicates a major role of membrane-associated amyloid oligomers in AD pathology. Our silica nanobowls (NB) coated with lipid-polymer have submicromolar affinity for Aβ binding. We demonstrate that NB scavenges distinct fractions of Aβs in a time-resolved manner from amyloid precursor protein-null neuronal cells after incubation with Aβ. At short incubation times in cell culture, NB-Aβ seeds have aggregation kinetics resembling that of extracellular fraction of Aβ, whereas at longer incubation times, NB-Aβ seeds scavenge membrane-associated Aβ. Aβ aggregates can be eluted from NB surfaces by mechanical agitation and appear to retain their aggregation driving domains as seen in seeding aggregation experiments. These results demonstrate that the NB system can be used for time-resolved separation of toxic Aβ species from biological samples for characterization and in diagnostics. Scavenging membrane-associated amyloids using lipid-functionalized NB without chemical manipulation has wide applications in the diagnosis and therapy of AD and other neurodegenerative diseases, cancer, and cardiovascular conditions.     

Identification and characterization of PlAlix,the Alix homologue from the Mediterranean sea urchin Paracentrotus lividus

The sea urchin provides a relatively simple and tractable system for analyzing the early stages of embryo development. Here, we use the sea urchin species, Paracentrotus lividus, to investigate the role of Alix in key stages of embryogenesis, namely the egg fertilization and the first cleavage division. Alix is a multifunctional protein involved in different cellular processes including endocytic membrane trafficking, filamentous (F)‐actin remodeling, and cytokinesis. Alix homologues have been identified in different metazoans; in these organisms, Alix is involved in oogenesis and in determination/differentiation events during embryo development. Herein, we describe the identification of the sea urchin homologue of Alix, PlAlix. The deduced amino acid sequence shows that Alix is highly conserved in sea urchins. Accordingly, we detect the PlAlix protein cross‐reacting with monoclonal Alix antibodies in extracts from P. lividus, at different developmental stages. Focusing on the role of PlAlix during early embryogenesis we found that PlAlix is a maternal protein that is expressed at increasingly higher levels from fertilization to the 2‐cell stage embryo. In sea urchin eggs, PlAlix localizes throughout the cytoplasm with a punctuated pattern and, soon after fertilization, accumulates in larger puncta in the cytosol, and in microvilli‐like protrusions. Together our data show that PlAlix is structurally conserved from sea urchin to mammals and may open new lines of inquiry into the role of Alix during the early stages of embryo development.     

Pretreatment of chemically-synthesized Aβ42 affects its biological activity in yeast

The tendency of amyloid β (Aβ₄₂) peptide to misfold and aggregate into insoluble amyloid fibrils in Alzheimer's disease (AD) has been well documented. Accumulation of Aβ₄₂ fibrils has been correlated with abnormal apoptosis and unscheduled cell division which can also trigger the death of neuronal cells, while oligomers can also exhibit similar activities. While investigations using chemically-synthesized Aβ₄₂ peptide have become common practice, there appear to be differences in outcomes from different preparations. In order to resolve this inconsistency, we report 2 separate methods of preparing chemically-synthesized Aβ₄₂ and we examined their effects in yeast. Hexafluoroisopropanol pretreatment caused toxicity while, ammonium hydroxide treated Aβ₄₂ induced cell proliferation in both C. glabrata and S. cerevisiae. The hexafluoroisopropanol prepared Aβ₄₂ had greater tendency to form amyloid on yeast cells as determined by thioflavin T staining followed by flow cytometry and microscopy. Both quiescent and non-quiescent cells were analyzed by these methods of peptide preparation. Non-quiescent cells were susceptible to the toxicity of Aβ₄₂ compared with quiescent cells (p < 0.005). These data explain the discrepancy in the previous publications about the effects of chemically-synthesized Aβ₄₂ on yeast cells. The effect of Aβ₄₂ on yeast cells was independent of the size of the peptide aggregates. However, the Aβ₄₂ pretreatment determined whether the molecular conformation of peptide resulted in proliferation or toxicity in yeast based assays.