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

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

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.     

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.     

Age-dependent high-density clustering of GM1 ganglioside at presynaptic neuritic terminals promotes amyloid beta-protein fibrillogenesis

The deposition of amyloid beta-protein (Abeta) is an invariable feature of Alzheimer's disease (AD); however, the biological mechanism underlying Abeta assembly into fibrils in the brain remains unclear. Here, we show that a high-density cluster of GM1 ganglioside (GM1), which was detected by the specific binding of a novel peptide (p3), appeared selectively on synaptosomes prepared from aged mouse brains. Notably, the synaptosomes bearing the high-density GM1 cluster showed extraordinary potency to induce Abeta assembly, which was suppressed by an antibody specific to GM1-bound Abeta, an endogenous seed for AD amyloid. Together with evidence that Abeta deposition starts at presynaptic terminals in the AD brain and that GM1 levels significantly increase in amyloid-positive synaptosomes prepared from the AD brain, our results suggest that the age-dependent high-density GM1 clustering at presynaptic neuritic terminals is a critical step for Abeta deposition in AD.     

In vitro and in vivo degradation of Abeta peptide by peptidases coupled to erythrocytes

It is generally believed that amyloid β peptides (Aβ) are the key mediators of Alzheimer's disease. Therapeutic interventions have been directed toward impairing the synthesis or accelerating the clearance of Aβ. An equilibrium between blood and brain Aβ exists mediated by carriers that transport Aβ across the blood–brain barrier. Passive immunotherapy has been shown to be effective in mouse models of AD, where the plasma borne antibody binds plasma Aβ causing an efflux of Aβ from the brain. As an alternative to passive immunotherapy we have considered the use of Aβ-degrading peptidases to lower plasma Aβ levels. Here we compare the ability of three Aβ-degrading peptidases to degrade Aβ. Biotinylated peptidases were coupled to the surface of biotinylated erythrocytes via streptavidin. These erythrocyte-bound peptidases degrade Aβ peptide in plasma. Thus, peptidases bound to or expressed on the surface of erythroid cells represent an alternative to passive immunotherapy.     

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

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

Microglial chemotaxis, activation, and phagocytosis of amyloid beta-peptide as linked phenomena in Alzheimer's disease

Microglia are widely held to play important pathophysiologic roles in Alzheimer's disease (AD). On exposure to amyloid β peptide (Aβ) they exhibit chemotactic, phagocytic, phenotypic and secretory responses consistent with scavenger cell activity in a localized inflammatory setting. Because AD microglial chemotaxis, phagocytosis, and secretory activity have common, tightly linked soluble intermediaries (e.g., cytokines, chemokines), cell surface intermediaries (e.g., receptors, opsonins), and stimuli (e.g., highly inert Aβ deposits and exposed neurofibrilly tangles), the mechanisms for microglial clearance of Aβ are necessarily coupled to localized inflammatory mechanisms that can be cytotoxic to nearby tissue. This presents a critical dilemma for strategies to remove Aβ by enhancing micoglial activation—a dilemma that warrants substantial further investigation.     

An N-terminal Fragment of the Prion Protein Binds to Amyloid-β Oligomers and Inhibits Their Neurotoxicity in Vivo

A hallmark of Alzheimer disease (AD) is the accumulation of the amyloid-β (Aβ) peptide in the brain. Considerable evidence suggests that soluble Aβ oligomers are responsible for the synaptic dysfunction and cognitive deficit observed in AD. However, the mechanism by which these oligomers exert their neurotoxic effect remains unknown. Recently, it was reported that Aβ oligomers bind to the cellular prion protein with high affinity. Here, we show that N1, the main physiological cleavage fragment of the cellular prion protein, is necessary and sufficient for binding early oligomeric intermediates during Aβ polymerization into amyloid fibrils. The ability of N1 to bind Aβ oligomers is influenced by positively charged residues in two sites (positions 23–31 and 95–105) and is dependent on the length of the sequence between them. Importantly, we also show that N1 strongly suppresses Aβ oligomer toxicity in cultured murine hippocampal neurons, in a Caenorhabditis elegans-based assay, and in vivo in a mouse model of Aβ-induced memory dysfunction. These data suggest that N1, or small peptides derived from it, could be potent inhibitors of Aβ oligomer toxicity and represent an entirely new class of therapeutic agents for AD.     

The influence of spin-labeled fluorene compounds on the assembly and toxicity of the aβ peptide

Background

The deposition and oligomerization of amyloid β (Aβ) peptide plays a key role in the pathogenesis of Alzheimer''s disease (AD). Aβ peptide arises from cleavage of the membrane-associated domain of the amyloid precursor protein (APP) by β and γ secretases. Several lines of evidence point to the soluble Aβ oligomer (AβO) as the primary neurotoxic species in the etiology of AD. Recently, we have demonstrated that a class of fluorene molecules specifically disrupts the AβO species.

Methodology/Principal Findings

To achieve a better understanding of the mechanism of action of this disruptive ability, we extend the application of electron paramagnetic resonance (EPR) spectroscopy of site-directed spin labels in the Aβ peptide to investigate the binding and influence of fluorene compounds on AβO structure and dynamics. In addition, we have synthesized a spin-labeled fluorene (SLF) containing a pyrroline nitroxide group that provides both increased cell protection against AβO toxicity and a route to directly observe the binding of the fluorene to the AβO assembly. We also evaluate the ability of fluorenes to target multiple pathological processes involved in the neurodegenerative cascade, such as their ability to block AβO toxicity, scavenge free radicals and diminish the formation of intracellular AβO species.

Conclusions

Fluorene modified with pyrroline nitroxide may be especially useful in counteracting Aβ peptide toxicity, because they posses both antioxidant properties and the ability to disrupt AβO species.     

Non-conjugated small molecule FRET for differentiating monomers from higher molecular weight amyloid beta species

Background

Systematic differentiation of amyloid (Aβ) species could be important for diagnosis of Alzheimer''s disease (AD). In spite of significant progress, controversies remain regarding which species are the primary contributors to the AD pathology, and which species could be used as the best biomarkers for its diagnosis. These controversies are partially caused by the lack of reliable methods to differentiate the complicated subtypes of Aβ species. Particularly, differentiation of Aβ monomers from toxic higher molecular weight species (HrMW) would be beneficial for drug screening, diagnosis, and molecular mechanism studies. However, fast and cheap methods for these specific aims are still lacking.

Principal Findings

We demonstrated the feasibility of a non-conjugated FRET (Förster resonance energy transfer) technique that utilized amyloid beta (Aβ) species as intrinsic platforms for the FRET pair assembly. Mixing two structurally similar curcumin derivatives that served as the small molecule FRET pair with Aβ40 aggregates resulted in a FRET signal, while no signal was detected when using Aβ40 monomer solution. Lastly, this FRET technique enabled us to quantify the concentrations of Aβ monomers and high molecular weight species in solution.

Significance

We believe that this FRET technique could potentially be used as a tool for screening for inhibitors of Aβ aggregation. We also suggest that this concept could be generalized to other misfolded proteins/peptides implicated in various pathologies including amyloid in diabetes, prion in bovine spongiform encephalopathy, tau protein in AD, and α-synuclein in Parkinson disease.     

Detection of Neuritic Plaques in Alzheimer's Disease Mouse Model

Alzheimer''s disease (AD) is the most common neurodegenerative disorder leading to dementia. Neuritic plaque formation is one of the pathological hallmarks of Alzheimer''s disease. The central component of neuritic plaques is a small filamentous protein called amyloid β protein (Aβ)¹, which is derived from sequential proteolytic cleavage of the beta-amyloid precursor protein (APP) by β-secretase and γ-secretase. The amyloid hypothesis entails that Aγ-containing plaques as the underlying toxic mechanism in AD pathology². The postmortem analysis of the presence of neuritic plaque confirms the diagnosis of AD. To further our understanding of Aγ neurobiology in AD pathogenesis, various mouse strains expressing AD-related mutations in the human APP genes were generated. Depending on the severity of the disease, these mice will develop neuritic plaques at different ages. These mice serve as invaluable tools for studying the pathogenesis and drug development that could affect the APP processing pathway and neuritic plaque formation. In this protocol, we employ an immunohistochemical method for specific detection of neuritic plaques in AD model mice. We will specifically discuss the preparation from extracting the half brain, paraformaldehyde fixation, cryosectioning, and two methods to detect neurotic plaques in AD transgenic mice: immunohistochemical detection using the ABC and DAB method and fluorescent detection using thiofalvin S staining method.     

Anesthetic propofol attenuates the isoflurane-induced caspase-3 activation and Aβ oligomerization

Accumulation and deposition of β-amyloid protein (Aβ) are the hallmark features of Alzheimer''s disease. The inhalation anesthetic isoflurane has been shown to induce caspase activation and increase Aβ accumulation. In addition, recent studies suggest that isoflurane may directly promote the formation of cytotoxic soluble Aβ oligomers, which are thought to be the key pathological species in AD. In contrast, propofol, the most commonly used intravenous anesthetic, has been reported to have neuroprotective effects. We therefore set out to compare the effects of isoflurane and propofol alone and in combination on caspase-3 activation and Aβ oligomerization in vitro and in vivo. Naïve and stably-transfected H4 human neuroglioma cells that express human amyloid precursor protein, the precursor for Aβ; neonatal mice; and conditioned cell culture media containing secreted human Aβ40 or Aβ42 were treated with isoflurane and/or propofol. Here we show for the first time that propofol can attenuate isoflurane-induced caspase-3 activation in cultured cells and in the brain tissues of neonatal mice. Furthermore, propofol-mediated caspase inhibition occurred when there were elevated levels of Aβ. Finally, isoflurane alone induces Aβ42, but not Aβ40, oligomerization, and propofol can inhibit the isoflurane-mediated oligomerization of Aβ42. These data suggest that propofol may mitigate the caspase-3 activation by attenuating the isoflurane-induced Aβ42 oligomerization. Our findings provide novel insights into the possible mechanisms of isoflurane-induced neurotoxicity that may aid in the development of strategies to minimize potential adverse effects associated with the administration of anesthetics to patients.     

Autophagic vacuoles are enriched in amyloid precursor protein-secretase activities: implications for beta-amyloid peptide over-production and localization in Alzheimer's disease

In Alzheimer’s disease (AD), the neuropathologic hallmarks of β-amyloid deposition and neurofibrillary degeneration are associated with early and progressive pathology of the endosomal–lysosomal system. Abnormalities of autophagy, a major pathway to lysosomes for protein and organelle turnover, include marked accumulations of autophagy-related vesicular compartments (autophagic vacuoles or AVs) in affected neurons. Here, we investigated the possibility that AVs contain the proteases and substrates necessary to cleave the amyloid precursor protein (APP) to Aβ peptide that forms β-amyloid, a key pathogenic factor in AD. AVs were highly purified using a well-established metrizamide gradient procedure from livers of transgenic YAC mice overexpressing wild-type human APP. By Western blot analysis, AVs contained APP, βCTF - the β-cleaved carboxyl-terminal domain of APP, and BACE, the protease-mediating β-cleavage of APP. β-Secretase activity measured against a fluorogenic peptide was significantly enriched in the AV fraction relative to whole-liver lysate. Compared to other recovered subcellular fractions, AVs exhibited the highest specific activity of γ-secretase based on a fluorogenic assay and inhibition by a specific inhibitor of γ-secretase, DAPT. AVs were also the most enriched subcellular fraction in levels of the γ-secretase components presenilin and nicastrin. Immunoelectron microscopy demonstrated selective immunogold labeling of AVs with antibodies specific for the carboxyl termini of human Aβ40 and Aβ42. These data indicate that AVs are a previously unrecognized and potentially highly active compartment for Aβ generation and suggest that the abnormal accumulation of AVs in affected neurons of the AD brain contributes to β-amyloid deposition.     

Anti-Aβ drug screening platform using human iPS cell-derived neurons for the treatment of Alzheimer's disease

Background

Alzheimer''s disease (AD) is a neurodegenerative disorder that causes progressive memory and cognitive decline during middle to late adult life. The AD brain is characterized by deposition of amyloid β peptide (Aβ), which is produced from amyloid precursor protein by β- and γ-secretase (presenilin complex)-mediated sequential cleavage. Induced pluripotent stem (iPS) cells potentially provide an opportunity to generate a human cell-based model of AD that would be crucial for drug discovery as well as for investigating mechanisms of the disease.

Methodology/Principal Findings

We differentiated human iPS (hiPS) cells into neuronal cells expressing the forebrain marker, Foxg1, and the neocortical markers, Cux1, Satb2, Ctip2, and Tbr1. The iPS cell-derived neuronal cells also expressed amyloid precursor protein, β-secretase, and γ-secretase components, and were capable of secreting Aβ into the conditioned media. Aβ production was inhibited by β-secretase inhibitor, γ-secretase inhibitor (GSI), and an NSAID; however, there were different susceptibilities to all three drugs between early and late differentiation stages. At the early differentiation stage, GSI treatment caused a fast increase at lower dose (Aβ surge) and drastic decline of Aβ production.

Conclusions/Significance

These results indicate that the hiPS cell-derived neuronal cells express functional β- and γ-secretases involved in Aβ production; however, anti-Aβ drug screening using these hiPS cell-derived neuronal cells requires sufficient neuronal differentiation.     

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.     

Differential Regulation of Amyloid-β Endocytic Trafficking and Lysosomal Degradation by Apolipoprotein E Isoforms

Aggregation of amyloid-β (Aβ) peptides leads to synaptic disruption and neurodegeneration in Alzheimer disease (AD). A major Aβ clearance pathway in the brain is cellular uptake and degradation. However, how Aβ traffics through the endocytic pathway and how AD risk factors regulate this event is unclear. Here we show that the majority of endocytosed Aβ in neurons traffics through early and late endosomes to the lysosomes for degradation. Overexpression of Rab5 or Rab7, small GTPases that function in vesicle fusion for early and late endosomes, respectively, significantly accelerates Aβ endocytic trafficking to the lysosomes. We also found that a portion of endocytosed Aβ traffics through Rab11-positive recycling vesicles. A blockage of this Aβ recycling pathway with a constitutively active Rab11 mutant significantly accelerates cellular Aβ accumulation. Inhibition of lysosomal enzymes results in Aβ accumulation and aggregation. Importantly, apolipoprotein E (apoE) accelerates neuronal Aβ uptake, lysosomal trafficking, and degradation in an isoform-dependent manner with apoE3 more efficiently facilitating Aβ trafficking and degradation than apoE4, a risk factor for AD. Taken together, our results demonstrate that Aβ endocytic trafficking to lysosomes for degradation is a major Aβ clearance pathway that is differentially regulated by apoE isoforms. A disturbance of this pathway can lead to accumulation and aggregation of cellular Aβ capable of causing neurotoxicity and seeding amyloid.     

MAP-2 immunolabeling can distinguish diffuse from dense-core amyloid plaques in brains with Alzheimer's disease

Alzheimer's disease (AD) neuropathology is characterized by the presence of diffuse and dense-core (neuritic) amyloid plaques in specific areas of the brain. The origin of these plaques and the relationship between them is poorly understood. Current methods to identify clearly these types of plaques in the AD brains are largely dependent upon morphological characteristics. Dense-core amyloid plaques in the entorhinal cortex and hippocampus of AD brains might arise from the lysis of neurons overburdened by excessive intracellular deposition of amyloid beta_1-42 (Aβ42) peptide. The local release of active lysosomal enzymes, which persist within these plaques, might degrade most of the released intracellular proteins, leaving behind only those that are resistant to proteolytic digestion, such as ubiquitin, tau, neurofilament proteins and amyloid. To test the possibility that proteins that are sensitive to proteolysis may be degraded selectively in plaques, we used immunohistochemistry to examine the distribution of microtubule-associated protein-2 (MAP-2), a protein localized primarily in neuronal dendrites and known to be sensitive to proteolysis. Uniform MAP-2 immunolabeling was detected throughout the somatodendritic compartment of neurons in age-matched control cortical brain tissues as well as throughout areas of Aβ42-positive diffuse plaques in AD brains. In contrast, analysis of serial sections revealed that MAP-2 was absent from Aβ42-positive dense-core plaques in AD brains. Our results indicate that this differential MAP-2 immunolabeling pattern among plaques may be employed as a reliable and sensitive method to distinguish dense-core plaques from diffuse plaques within AD brain tissue. Furthermore, this biochemical distinction indicates that dense-core and diffuse plaques are formed by different mechanisms.     

Hematopoietic CC-chemokine receptor 2 (CCR2) competent cells are protective for the cognitive impairments and amyloid pathology in a transgenic mouse model of Alzheimer's disease

Monocytes emigrate from bone marrow, can infiltrate into brain, differentiate into microglia and clear amyloid β (Aβ) from the brain of mouse models of Alzheimer’s disease (AD). Here we show that these mechanisms specifically require CC-chemokine receptor 2 (CCR2) expression in bone marrow cells (BMCs). Disease progression was exacerbated in APP_Swe/PS1 mice (transgenic mice expressing a chimeric amyloid precursor protein [APPSwe] and human presenilin 1 [PS1]) harboring CCR2-deficient BMCs. Indeed, transplantation of CCR2-deficient BMCs enhanced the mnesic deficit and increased the amount of soluble Aβ and expression of transforming growth factor (TGF)-β1 and TGF-β receptors. By contrast, transplantation of wild-type bone marrow stem cells restored memory capacities and diminished soluble Aβ accumulation in APP_Swe/PS1 and APP_Swe/PS1/CCR2^−/− mice. Finally, gene therapy using a lentivirus-expressing CCR2 transgene in BMCs prevented cognitive decline in this mouse model of AD. Injection of CCR2 lentiviruses restored CCR2 expression and functions in monocytes. The presence of these cells in the brain of non-irradiated APP_Swe/PS1/CCR2^−/− mice supports the concept that they can be used as gene vehicles for AD. Decreased CCR2 expression in bone marrow–derived microglia may therefore play a major role in the etiology of this neurodegenerative disease.     

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.     

A peptide that binds specifically to the β-amyloid of Alzheimer's disease: selection and assessment of anti-β-amyloid neurotoxic effects

The accumulation of the amyloid-β peptide (Aβ) into amyloid plaques, an essential event in Alzheimer''s disease (AD) pathogenesis, has caused researchers to seek compounds that physiologically bind Aβ and modulate its aggregation and neurotoxicity. In order to develop new Aβ-specific peptides for AD, a randomized 12-mer peptide library with Aβ_1-10 as the target was used to identify peptides in the present study. After three rounds of selection, specific phages were screened, and their binding affinities to Aβ_1-10 were found to be highly specific. Finally, a special peptide was synthesized according to the sequences of the selected phages. In addition, the effects of the special peptide on Aβ aggregation and Aβ-mediated neurotoxicity in vitro and in vivo were assessed. The results show that the special peptide not only inhibited the aggregation of Aβ into plaques, but it also alleviated Aβ-induced PC12 cell viability and apoptosis at appropriate concentrations as assessed by the cell counting kit-8 assay and propidium iodide staining. Moreover, the special peptide exhibited a protective effect against Aβ-induced learning and memory deficits in rats, as determined by the Morris water maze task. In conclusion, we selected a peptide that specifically binds Aβ_1-10 and can modulate Aβ aggregation and Aβ-induced neuronal damage. This opens up possibilities for the development of a novel therapeutic approach for the treatment of AD.