Aβ Oligomer-induced Neurotoxicity Research Articles

A Novel Targeted and High-Efficiency Nanosystem for Combinational Therapy for Alzheimer's Disease.

Alzheimer's disease (AD) remains the most prevalent neurodegenerative disease, and no effective treatment is available yet. Metal‐ion‐triggered aggregates of amyloid‐beta (Aβ) peptide and acetylcholine imbalance are reported to be possible factors in AD pathogenesis. Thus, a combination therapy that can not only inhibit and reduce Aβ aggregation but also simultaneously regulate acetylcholine imbalance that can serve as a potential treatment for AD is needed. Here, clioquinol (metal‐ion chelating agent) and donepezil (acetylcholinesterase (AChE) inhibitor) co‐encapsulated human serum albumin (HSA) nanoparticles (dcHGT NPs) are designed, which are modified with transcriptional activator protein (TAT) and monosialotetrahexosylganglioside (GM1). The GM1 lipid and TAT peptide endow this drug delivery nanosystem with high brain entry efficiency and long‐term retention capabilities through intranasal administration. It is found that dcHGT NPs can significantly inhibit and eliminate Aβ aggregation, relieve acetylcholine‐related inflammation in microglial cells, and protect primary neurons from Aβ oligomer‐induced neurotoxicity in vitro. The alleviation of Aβ‐related inflammation and AChE‐inhibited effect further synergistically adjust acetylcholine imbalance. It is further demonstrated that dcHGT NPs reduce Aβ deposition, ameliorate neuron morphological changes, rescue memory deficits, and greatly improve acetylcholine regulation ability in vivo. This multifunctional synergetic nanosystem can be a new candidate to achieve highly efficient combination therapy for AD.

<p>Transferrin-Modified Osthole PEGylated Liposomes Travel the Blood-Brain Barrier and Mitigate Alzheimer’s Disease-Related Pathology in APP/PS-1 Mice</p>

IntroductionOsthole (Ost) is a coumarin compound that strengthens hippocampal neurons and neural stem cells against Aβ oligomer-induced neurotoxicity in mice, and is a potential drug for the treatment of Alzheimer's disease (AD). However, the effectiveness of the drug is limited by its solubility and bioavailability, as well as by the low permeability of the blood-brain barrier (BBB). In this study, a kind of transferrin-modified Ost liposomes (Tf-Ost-Lip) was constructed, which could improve the bioavailability and enhance brain targeting.MethodsTf-Ost-Lip was prepared by thin-film hydration method. The ability of liposomal formulations to translocate across BBB was investigated using in vitro BBB model. And the protective effect of Tf-Ost-Lip was evaluated in APP-SH-SY5Y cells. In addition, we performed pharmacokinetics study and brain tissue distribution analysis of liposomal formulations in vivo. We also observed the neuroprotective effect of the varying formulations in APP/PS-1 mice.ResultsIn vitro studies reveal that Tf-Ost-Lip could increase the intracellular uptake of hCMEC/D3 cells and APP-SH-SY5Y cells, and increase the drug concentration across the BBB. Additionally, Tf-Ost-Lip was found to exert a protective effect on APP-SH-SY5Y cells. In vivo studies of pharmacokinetics and the Ost distribution in brain tissue indicate that Tf-Ost-Lip prolonged the cycle time in mice and increased the accumulation of Ost in the brain. Furthermore, Tf-Ost-Lip was also found to enhance the effect of Ost on the alleviation of Alzheimer’s disease-related pathology.ConclusionTransferrin-modified liposomes for delivery of Ost has great potential for AD treatment.

Targeting Hsc70-based autophagy to eliminate amyloid β oligomers

Amyloid β (Aβ) oligomers may be a real culprit in the pathogenesis of Alzheimer’s disease (AD); therefore, the elimination of these toxic oligomers may be of great significance for AD therapy. Autophagy is the catabolic process by which lysosomes degrade cytosolic components, and heat shock cognate 70 kDa protein (Hsc70) binds to proteins with their KFERQ-like motifs [also known as chaperone-mediated autophagy (CMA) motifs] and carries them to lysosomes through CMA or late endosomes through endosomal microautophagy (eMI) for degradation. In this study, our strategy is to make the pathological Aβ become one selective and suitable substrate for CMA and eMI (termed as Hsc70-based autophagy) by tagging its oligomers with multiple CMA motifs. First, we design and synthesize Aβ oligomer binding peptides with three CMA motifs. Second, we determine that the peptide can help Aβ oligomers enter endosomes and lysosomes, which can be further enhanced by ketone. More importantly, we find that the peptide can dramatically reduce Aβ oligomers in induced pluripotent stem cell (iPSC) cortical neurons derived from AD patient fibroblasts and protect primary cultured cortical neurons against the Aβ oligomer-induced neurotoxicity. In conclusion, we demonstrate that the peptide targeting Hsc70-based autophagy can effectively eliminate Aβ oligomers and have superior neuroprotective activity.

PROTECTIVE PROPERTY OF P3-ALCβ AGAINST A NEUROTOXICITY INDUCED BY Aβ OLIGOMER

Neuronal p3-Alcβ peptide is generated from the precursor protein Alcadeinβ (Alcβ) by a cleavage of α- and γ-secretases. Alcβ generates the C-terminal variant peptides including a major p3-Alcβ37, as does APP. It is understood that Aβ42 level tends to decrease in AD patients in cerebrospinal fluid (CSF) by deposition of Aβ aggregate in brain compared to age-matched non-demented subjects. Although, p3-Alcβ levels are comparable to those of Aβ levels, the trend of non-aggregatable p3-Alcβ levels in CSF of AD subjects has not been analyzed. Moreover, function of p3-Alcβ remains unclear. In this study, we analyzed the change of p3-Alcβ in CSF of AD and explored the function of p3-Alcβ, especially in the role to prevent Aβ oligomer-induced neurotoxicity. We developed a new sandwich ELISA to quantify p3-Alcβ37. The primary cultures of mixed mouse cortical and hippocampal neurons were used to assess functions of p3-Alcβ in Aβ oligomer-induced neurotoxicity. Partial sequence peptides of p3-Alcβ were examined to identify functional regions of p3-Alcβ in vivo and in vitro. To analyze pharmacokinetics of the short peptide including functional amino acid sequence in mouse, we developed another sELISA. CSF p3-Alcβ level significantly decreased in Alzheimer's disease (AD) and/or mild cognitive impairment, compared to age-matched non-demented subjects (see Poster by Hata et al). Interestingly, a synthetic p3-Alcβ37 preserved primary cultured neurons from an impairment by Aβ42 oligomers in dose-dependent manner. Independent study identified the functional amino acid sequence in p3-Alcβ, which restored a cognitive impairment induced by Aβ oligomers in mouse. To develop a new drug therapy with this short peptide/a lead peptide including the functional amino acid sequence, we examined levels of the lead peptide in blood and CSF with sELISA. Following the subcutaneous administration of the lead peptide into mice, the peptide was detected in blood quickly and followed in CSF, indicating the brain transport of the peptide by a peripheral administration. The p3-Alcβ shows a protective effect against neurotoxicity induced by Aβ oligomer. Shorter peptide of p3-Alcβ is a drugable lead with an ability of brain transport by peripheral administration.

Fucoxanthin, a Marine Carotenoid, Attenuates β-Amyloid Oligomer-Induced Neurotoxicity Possibly via Regulating the PI3K/Akt and the ERK Pathways in SH-SY5Y Cells.

Alzheimer's disease (AD), the most common neurodegenerative disorder, is characterized by neurofibrillary tangles, synaptic impairments, and loss of neurons. Oligomers of β-amyloid (Aβ) are widely accepted as the main neurotoxins to induce oxidative stress and neuronal loss in AD. In this study, we discovered that fucoxanthin, a marine carotenoid with antioxidative stress properties, concentration dependently prevented Aβ oligomer-induced increase of neuronal apoptosis and intracellular reactive oxygen species in SH-SY5Y cells. Aβ oligomers inhibited the prosurvival phosphoinositide 3-kinase (PI3K)/Akt cascade and activated the proapoptotic extracellular signal-regulated kinase (ERK) pathway. Moreover, inhibitors of glycogen synthase kinase 3β (GSK3β) and mitogen-activated protein kinase (MEK) synergistically prevented Aβ oligomer-induced neuronal death, suggesting that the PI3K/Akt and ERK pathways might be involved in Aβ oligomer-induced neurotoxicity. Pretreatment with fucoxanthin significantly prevented Aβ oligomer-induced alteration of the PI3K/Akt and ERK pathways. Furthermore, LY294002 and wortmannin, two PI3K inhibitors, abolished the neuroprotective effects of fucoxanthin against Aβ oligomer-induced neurotoxicity. These results suggested that fucoxanthin might prevent Aβ oligomer-induced neuronal loss and oxidative stress via the activation of the PI3K/Akt cascade as well as inhibition of the ERK pathway, indicating that further studies of fucoxanthin and related compounds might lead to a useful treatment of AD.

Optimized-SopungSunkiwon, a Herbal Formula, Attenuates Aβ Oligomer-Induced Neurotoxicity in Alzheimer's Disease Models.

Alzheimer's disease (AD), the most common form of dementia, is an age-related neurodegenerative disease that is characterized by memory dysfunction, neuronal cell damage, and neuroinflammation. It is believed that AD-related pathology is mostly due to the overproduction of Aβ, especially the oligomeric form (AβO), in the brain. Evidence of the effects of multifunctional medicinal herbs in the treatment of AD has been steadily increasing. Optimized-SopungSunkiwon (OSS), a multiherbal formulation that is composed of six medicinal herbs derived from SopungSunkiwon, is a traditional medicine that is prescribed for neurodegenerative disorders in elderly patients. We previously reported that OSS showed an antiamnesic and memory enhancing effect in mice, but it is unknown whether OSS has a protective effect against AβO neurotoxicity. In this study, we investigated the effects of OSS in AD models induced by AβO in vitro and in vivo. We found that OSS protected neuronal cells and inhibited the generation of nitric oxide and reactive oxygen species against AβO toxicity in vitro. These results were confirmed by in vivo data that oral administration of OSS for 14 days attenuated memory impairments and neuronal cell death by modulating gliosis, glutathione depletion, and synaptic damage in the mouse hippocampus induced by AβO.

Atorvastatin prevents Aβ oligomer-induced neurotoxicity in cultured rat hippocampal neurons by inhibiting Tau cleavage

The proteolytic cleavage of Tau is involved in Aβ-induced neuronal dysfunction and cell death. In this study, we investigated whether atorvastatin could prevent Tau cleavage and hence prevent Aβ1-42 oligomer (AβO)-induced neurotoxicity in cultured cortical neurons. Cultured rat hippocampal neurons were incubated in the presence of AβOs (1.25 μmol/L) with or without atorvastatin pretreatment. ATP content and LDH in the culture medium were measured to assess the neuronal viability. Caspase-3/7 and calpain protease activities were detected. The levels of phospho-Akt, phospho-Erk1/2, phospho-GSK3β, p35 and Tau proteins were measured using Western blotting. Treatment of the neurons with AβO significantly decreased the neuronal viability, induced rapid activation of calpain and caspase-3/7 proteases, accompanied by Tau degradation and relatively stable fragments generated in the neurons. AβO also suppressed Akt and Erk1/2 kinase activity, while increased GSK3β and Cdk5 activity in the neurons. Pretreatment with atorvastatin (0.5, 1, 2.5 μmol/L) dose-dependently inhibited AβO-induced activation of calpain and caspase-3/7 proteases, and effectively diminished the generation of Tau fragments, attenuated synaptic damage and increased neuronal survival. Atorvastatin pretreatment also prevented AβO-induced decreases in Akt and Erk1/2 kinase activity and the increases in GSK3β and Cdk5 kinase activity. Atorvastatin prevents AβO-induced neurotoxicity in cultured rat hippocampal neurons by inhibiting calpain- and caspase-mediated Tau cleavage.

Protective effects of glycycoumarin and procyanidin B1, active components of traditional Japanese medicine yokukansan, on amyloid β oligomer-induced neuronal death

Ethnopharmacological relevanceYokukansan, a traditional Japanese (Kampo) medicine, is composed of seven medicinal herbs, and has been traditionally used to treat neurosis, insomnia, and night crying and irritability in children. Yokukansan and its constituent herbs, Glycyrrhiza and Uncaria Hook, have recently been shown to have protective effects against amyloid β (Aβ) oligomer-induced apoptosis by suppressing the activation of caspase-3 in primary cultured neurons. The aim of the present study was to identify the effective components of Glycyrrhiza and Uncaria Hook against Aβ oligomer-induced neurotoxicity. We also attempted to clarify the mechanisms by which yokukansan and these herbs, as well as their components, suppressed the activation of caspase-3 in Aβ oligomer-treated neurons. Materials and methodsRat primary cultured cortical neurons were treated with Aβ oligomer (3μM). The protective effects of 16 components derived from Glycyrrhiza or Uncaria Hook against Aβ oligomer-induced neurotoxicity were determined using the MTT reduction assay 48h after the treatment. The suppressive effects of the test substances, i.e., yokukansan, Glycyrrhiza, Uncaria Hook, and screened components, on the Aβ oligomer-induced activation of caspase-3(/7) were evaluated using the caspase-Glo assay 48h after the Aβ oligomer treatment. The suppressive effects of the test substances on the activation of caspase-8 and -9, both of which are located upstream of caspase-3, were also examined 24h after the Aβ oligomer treatment. ResultsTwo of the 16 components tested, glycycoumarin derived from Glycyrrhiza and procyanidin B1 derived from Uncaria Hook, significantly inhibited Aβ oligomer-induced neuronal death in a dose-dependent manner. Glycyrrhiza, Uncaria Hook, and yokukansan significantly suppressed the Aβ oligomer-induced activation of caspase-3 as well as caspase-8 and -9. Glycycoumarin also suppressed the activation of caspase-3, but not caspase-8 and -9. Procyanidin B1 suppressed the activation of caspase-3, -8, and -9. ConclusionsOur results demonstrated that glycycoumarin and procyanidin B1 had ameliorative effects on Aβ oligomer-induced neurotoxicity. The neuroprotective effects of glycycoumarin are thought to be due to the attenuated activation of caspase-3, but not caspase-8 or -9. Procyanidin B1, as well as yokukansan, Glycyrrhiza, and Uncaria Hook, may attenuate the activation of caspase-3 by inhibiting that of caspase-8 and -9.

Glycyrrhiza and Uncaria Hook contribute to protective effect of traditional Japanese medicine yokukansan against amyloid β oligomer-induced neuronal death

Ethnopharmacological relevanceYokukansan, a traditional Japanese (Kampo) medicine, composed of seven medicinal herbs has been traditionally used to treat neurosis, insomnia, and night crying and irritability in children. Recently, this medicine has been reported to improve the behavioral and psychological symptoms of dementia (BPSD) that often become problematic in patients with Alzheimer's disease (AD). Aim of the studyAmyloid β (Aβ) oligomers, which are extremely toxic to neurons, are involved in neurodegeneration in AD. In animals, yokukansan has been proven to improve memory impairments and BPSD-like behavior in transgenic mice overexpressing amyloid precursor protein and mice intracerebroventricularly injected with Aβ oligomers. These results suggest that yokukansan is potentially able to reduce the neurotoxicity of Aβ oligomers. Therefore, the present study aimed to explore the improving effects brought by yokukansan that consists of seven herbs for Aβ oligomer-induced neurotoxicity in vitro and to identify the candidate herbs in yokukansan's action. Materials and methodsPrimary cultured rat cortical neurons were used. Neurotoxicity induced by Aβ oligomers (3µM) and improving effects of yokukansan (300–1000µg/mL) and its constituent herbs were evaluated in MTT assay, DNA fragmentation analysis, and electron microscopic analysis at 48h after treatment with Aβ oligomers and drugs. Moreover, changes in expression of genes related to endoplasmic reticulum (ER) stress and in caspase-3 activity that is the enzyme closely related to apoptosis were analyzed to investigate the underlying mechanisms. ResultsYokukansan ameliorated Aβ oligomer-induced neuronal damage in a dose-dependent manner in the MTT assay. This drug also suppressed DNA fragmentation caused by Aβ oligomers. Electron microscopic analysis suggested that yokukansan reduced karyopyknosis and the expansion of rough ER caused by Aβ oligomers. However, neither Aβ oligomers nor yokukansan affected the mRNA expression of any ER stress-related genes, including CHOP and GRP78. On the other hand, yokukansan dose-dependently suppressed Aβ oligomer-induced activation of caspase-3. Among the seven constituents of yokukansan, Glycyrrhiza and Uncaria Hook (60–200µg/mL) suppressed Aβ oligomer-induced neuronal damage, DNA fragmentation, karyopyknosis, and caspase-3 activation to almost the same extent as yokukansan. ConclusionsThe present results suggest that yokukansan possesses an ameliorative effect against Aβ oligomer-induced neuronal apoptosis through the suppression of caspase-3 activation. Glycyrrhiza and Uncaria Hook may, at least in part, contribute to the neuroprotective effect of yokukansan. These mechanisms may underlie the improving effects of yokukansan on memory impairment and BPSD-like behaviors induced by Aβ oligomers.

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, Aβ1–42 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 Aβ1–42 oligomers for 1h. Further analysis revealed that EphB2 overexpression increased synaptic NR1 and NR2B expression in Aβ1–42 oligomer-treated neurons. Moreover, EphB2 overexpression prevented Aβ1–42 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 Aβ1–42 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.

Cellular prion protein is essential for oligomeric amyloid- -induced neuronal cell death

In Alzheimer disease (AD), amyloid-β (Aβ) oligomer is suggested to play a critical role in imitating neurodegeneration, although its pathogenic mechanism remains to be determined. Recently, the cellular prion protein (PrP(C)) has been reported to be an essential co-factor in mediating the neurotoxic effect of Aβ oligomer. However, these previous studies focused on the synaptic plasticity in either the presence or the absence of PrP(C) and no study to date has reported whether PrP(C) is required for the neuronal cell death, the most critical element of neurodegeneration in AD. Here, we show that Prnp(-/-) mice are resistant to the neurotoxic effect of Aβ oligomer in vivo and in vitro. Furthermore, application of an anti-PrP(C) antibody or PrP(C) peptide prevents Aβ oligomer-induced neurotoxicity. These findings are the first to demonstrate that PrP(C) is required for Aβ oligomer-induced neuronal cell death, the pathology essential to cognitive loss.

Prevention of Amyloid-Beta Oligomer-Induced Neuronal Death by EGTA, Estradiol, and Endocytosis Inhibitor

BACKGROUND AND OBJECTIVE. Alzheimer's disease is a progressive neurodegenerative disease that is biochemically characterized by the accumulation of amyloid beta (Aβ) peptides in the brain. The current hypothesis suggests that Aβ oligomers rather than fibrillar aggregates are the most toxic species of Aβ though the mechanisms of their neurotoxicity are unclear. The authors have previously shown that small Aβ(1-42) oligomers at around 1 µM concentration caused rapid (in 24 h) neuronal death in cerebellar granule cell (CGC) cultures. In this study, we aimed to investigate whether protracted (up to 7 days) incubation of CGC cultures with lower submicromolar concentration of various aggregates of Aβ(1-42) had an effect on viability of neurons. In order to get some insight into the mechanism of Aβ-induced cell death, we also sought to determine whether extracellular Ca(2+) and process of endocytosis contributed to Aβ oligomer-induced neurotoxicity and whether pharmacological interventions into these processes would prevent Aβ oligomer-induced cell death. MATERIAL AND METHODS. Primary cultures of CGC were treated with various aggregate forms of Aβ(1-42). Cell viability was assessed by fluorescent microscopy using propidium iodide and Hoechst 33342 staining. RESULTS. Exposure of neurons to 500 nM Aβ(1-42) oligomers for 72-168 h caused extensive neuronal necrosis. Lower concentrations (100-250 nM) were not toxic to cells during 7 days of incubation. Aβ(1-42) monomers and fibrils had no effect on neuronal viability even after 7 days of incubation. Treatment of neurons with EGTA, steroid hormone 17β-estradiol, and methyl-β-cyclodextrin significantly reduced Aβ(1-42) oligomers-induced neuronal death. CONCLUSIONS. The results show that submicromolar concentrations of Aβ(1-42) oligomers were highly toxic to neurons during protracted incubation inducing neuronal necrosis that can be prevented by chelating extracellular Ca(2+) with EGTA, inhibiting endocytosis with methyl-β-cyclodextrin, or by estradiol, which may protect against mitochondrial permeability transition pore opening.

ERK1/2 Activation Mediates Aβ Oligomer-induced Neurotoxicity via Caspase-3 Activation and Tau Cleavage in Rat Organotypic Hippocampal Slice Cultures

In this study, we investigated the molecular basis for the altered signal transduction associated with soluble amyloid beta-protein (Abeta) oligomer-mediated neurotoxicity in the hippocampus, which is primarily linked to cognitive dysfunction in Alzheimer disease (AD). As measured by media lactate dehydrogenase levels, and staining with propidium iodide, acute exposure to low micromolar concentrations of the Abeta1-42 oligomer significantly induced cell death. This was accompanied by activation of the ERK1/2 signal transduction pathway in rat organotypic hippocampal slices. Notably, this resulted in caspase-3 activation by a process that led to proteolytic cleavage of Tau, which was recently confirmed to occur in AD brains. Tau cleavage likely occurred in the absence of overt synaptic loss, as suggested by the preserved levels of synaptophysin, a presynaptic marker. Moreover, among the pharmacological agents tested to inhibit several kinase cascades, only the ERK inhibitor significantly attenuated Abeta1-42 oligomer-induced toxicity concomitant with the reduction of activation of ERK1/2 and caspase-3 to a lesser extent. Importantly, the caspase-3 inhibitor also decreased Abeta oligomer-induced cell death, with no appreciable effect on the ERK signaling pathway, although such treatment was effective in reducing caspase-3 activation and Tau cleavage. Therefore, these results suggest that local targeting of the ERK1/2 signaling pathway to reduce Tau cleavage, as occurs with the inhibition of caspase-3 activation, may modulate the neurotoxic effects of soluble Abeta oligomer in the hippocampus and provide the rationale for symptomatic treatment of AD.