WNT5A signaling contributes to Aβ-induced neuroinflammation and neurotoxicity

Neurodegenration is a pathological hallmark of Alzheimer's disease (AD), but the underlying molecular mechanism remains elusive. Here, we present evidence that reveals a crucial role of Wnt5a signaling in this process. We showed that Wnt5a and its receptor Frizzled-5 (Fz5) were up-regulated in the AD mouse brain, and that beta-amyloid peptide (Aβ), a major constituent of amyloid plaques, stimulated Wnt5a and Fz5 expression in primary cortical cultures; these observations indicate that Wnt5a signaling could be aberrantly activated during AD pathogenesis. In support of such a possibility, we observed that inhibition of Wnt5a signaling attenuated while activation of Wnt5a signaling enhanced Aβ-evoked neurotoxicity, suggesting a role of Wnt5a signaling in AD-related neurodegeneration. Furthermore, we also demonstrated that Aβ-induced neurotoxicity depends on inflammatory processes, and that activation of Wnt5a signaling elicited the expression of proinflammatory cytokines IL-1β and TNF-α whereas inhibition of Wnt5a signaling attenuated the Aβ-induced expression of the cytokines in cortical cultures. Our findings collectively suggest that aberrantly up-regulated Wnt5a signaling is a crucial pathological step that contributes to AD-related neurodegeneration by regulating neuroinflammation.     

Diammonium glycyrrhizinate upregulates PGC-1α and protects against Aβ1-42-induced neurotoxicity

Mitochondrial dysfunction is a hallmark of beta-amyloid (Aβ)-induced neurotoxicity in Alzheimer's disease (AD), and is considered an early event in AD pathology. Diammonium glycyrrhizinate (DG), the salt form of Glycyrrhizin, is known for its anti-inflammatory effects, resistance to biologic oxidation and membranous protection. In the present study, the neuroprotective effects of DG on Aβ(1-42)-induced toxicity and its potential mechanisms in primary cortical neurons were investigated. Exposure of neurons to 2 μM Aβ(1-42) resulted in significant viability loss and cell apoptosis. Accumulation of reactive oxygen species (ROS), decreased mitochondrial membrane potential, and activation of caspase-9 and caspase-3 were also observed after Aβ(1-42) exposure. All these effects induced by Aβ(1-42) were markedly reversed by DG treatment. In addition, DG could alleviate lipid peroxidation and partially restore the mitochondrial function in Aβ(1-42)-induced AD mice. DG also significantly increased the PGC-1α expression in vivo and in vitro, while knocking down PGC-1α partially blocked the protective effects, which indicated that PGC-1α contributed to the neuroprotective effects of DG. Furthermore, DG significantly decreased the escape latency and search distance and increased the target crossing times of Aβ(1-42)-induced AD mice in the Morris water maze test. Therefore, these results demonstrated that DG could attenuate Aβ(1-42)-induced neuronal injury by preventing mitochondrial dysfunction and oxidative stress and improved cognitive impairment in Aβ(1-42)-induced AD mice, indicating that DG exerted potential beneficial effects on AD.     

‘Click’ assembly of glycoclusters and discovery of a trehalose analogue that retards Aβ40 aggregation and inhibits Aβ40-induced neurotoxicity

Osmolytes have been proposed as treatments for neurodegenerative proteinopathies including Alzheimer’s disease. However, for osmolytes to reach the clinic their efficacy must be improved. In this work, copper(I)-catalyzed azide–alkyne cycloaddition chemistry was used to synthesize glycoclusters bearing six copies of trehalose, lactose, galactose or glucose, with the aim of improving the potency of these osmolytes via multivalency. A trehalose glycocluster was found to be superior to monomeric trehalose in its ability to retard the formation of amyloid-beta peptide 40 (Aβ40) fibrils and protect neurons from Aβ40-induced cell death.     

Human apolipoprotein A–I binds amyloid-β and prevents Aβ-induced neurotoxicity

Aggregates of the amyloid-β peptide (Aβ) play a central role in the pathogenesis of Alzheimer's disease (AD). Identification of proteins that physiologically bind Aβ and modulate its aggregation and neurotoxicity could lead to the development of novel disease-modifying approaches in AD. By screening a phage display peptide library for high affinity ligands of aggregated Aβ_1–42, we isolated a peptide homologous to a highly conserved amino acid sequence present in the N-terminus of apolipoprotein A–I (apoA-I). We show that purified human apoA-I and Aβ form non-covalent complexes and that interaction with apoA-I affects the morphology of amyloid aggregates formed by Aβ. Significantly, Aβ/apoA-I complexes were also detected in cerebrospinal fluid from AD patients. Interestingly, apoA-I and apoA-I-containing reconstituted high density lipoprotein particles protect hippocampal neuronal cultures from Aβ-induced oxidative stress and neurodegeneration. These results suggest that human apoA-I modulates Aβ aggregation and Aβ-induced neuronal damage and that the Aβ-binding domain in apoA-I may constitute a novel framework for the design of inhibitors of Aβ toxicity.     

Insulin-degrading enzyme antagonizes insulin-dependent tissue growth and Aβ-induced neurotoxicity in Drosophila

Insulin-degrading enzyme (IDE) is implicated in the pathogenesis of type 2 diabetes mellitus (DM2) and Alzheimer’s disease (AD). Here we provide genetic evidence that Drosophila Ide (dIde) antagonizes the insulin signaling pathway and human Aβ-induced neurotoxicity in Drosophila. In this study, we also generated a dIde knockout mutant (dIde^KO) by gene targeting, and found that loss of IDE increases the content of the major insect blood sugar, trehalose, thus suggesting a conserved role of IDE in sugar metabolism. Using dIde^KO as a model, further investigations into the biological functions of IDE and its role in the pathogenesis of DM2 and AD can be made.     

Cannabidiol reduces Aβ-induced neuroinflammation and promotes hippocampal neurogenesis through PPARγ involvement

Peroxisome proliferator-activated receptor-γ (PPARγ) has been reported to be involved in the etiology of pathological features of Alzheimer's disease (AD). Cannabidiol (CBD), a Cannabis derivative devoid of psychomimetic effects, has attracted much attention because of its promising neuroprotective properties in rat AD models, even though the mechanism responsible for such actions remains unknown. This study was aimed at exploring whether CBD effects could be subordinate to its activity at PPARγ, which has been recently indicated as its putative binding site. CBD actions on β-amyloid-induced neurotoxicity in rat AD models, either in presence or absence of PPAR antagonists were investigated. Results showed that the blockade of PPARγ was able to significantly blunt CBD effects on reactive gliosis and subsequently on neuronal damage. Moreover, due to its interaction at PPARγ, CBD was observed to stimulate hippocampal neurogenesis. All these findings report the inescapable role of this receptor in mediating CBD actions, here reported.     

Cataract-linked mutation R188H promotes βB2-crystallin aggregation and fibrillization during acid denaturation

Cataract is characterized by the formation of light-scattering protein aggregates in the lens. β/γ-Crystallins are the predominant structural proteins in the cytosol of lens fiber cells, and more than fifty β/γ-crystallin mutations have been linked to autosomal dominant congenital cataract. However, the structural role of these mutations in the formation of the core structures of amorphous aggregates or amyloid-like fibrils has not been elucidated yet. In this research, we studied the effects of the V187M and R188H mutations on the aggregation and fibrillization of βB2-crystallin during acid denaturation. The behavior of V187M was the same as the WT protein, suggesting that the residue at position 187 contributed little to the aggregation/fibrillization process. R188H promoted the formation of amorphous aggregates at pH above 3 and accelerated fibrillization at pH 3. The distinct behaviors of the mutants suggested that the residue at position 188 might play a regulatory role in βB2-crystallin aggregation/fibrillization but not reside in the core of the aggregates/fibrils.     

Site-specific aspartic acid isomerization regulates self-assembly and neurotoxicity of amyloid-β

Amyloid-β (Aβ) proteins, which consist of 42 amino acids (Aβ_1–42), are the major constituent of neuritic plaques that form in the brains of senile patients with Alzheimer’s disease (AD). Several reports state that three aspartic acid (Asp) residues at positions 1, 7, and 23 in Aβ_1–42 in the plaques of patients with AD are highly isomerized from the l- to d-form. Using biophysical experiments, the present study shows that simultaneous d-isomerization of Asp residues at positions 7 and 23 (d-Asp^7,23) enhances oligomerization, fibril formation, and neurotoxic effect of Aβ_1–42. In addition, d-isomerization of Asp at position 1 (d-Asp¹) suppresses malignant effects induced by d-Asp^7,23 of Aβ_1–42. These results provide fundamental information to elucidate molecular mechanisms of AD pathogenesis and to develop potent inhibitors of amyloid aggregates and Aβ neurotoxicity.     

Attenuation of Aβ-induced neurotoxicity by thymoquinone via inhibition of mitochondrial dysfunction and oxidative stress

Beta-amyloid (Aβ) peptides are considered to play a major role in the pathogenesis of Alzheimer's disease (AD) and compounds that can prevent pathways of Aβ-induced neurotoxicity may be potential therapeutic agents for treatment of AD. This study examined the hypothesis that thymoquinone (TQ) would reduce oxidative stress and mitochondrial dysfunction in differentiated pheochromocytoma (PC 12) cells exposed to Aβ fragment 25-35 (Aβ(25-35)). To test this hypothesis, Aβ was used to induce an in vitro model of AD in differentiated PC 12 cell line of rat. After 24?h of exposure with Aβ(25-35), a significant reduction in cell viability and mitochondrial membrane potential (MMP) was observed. In addition, a significant elevation in the TBARS content and nitric oxide (NO) and activity of acetylcholine esterase (AChE) was observed which was restored significantly by TQ pretreatment. Furthermore, TQ also ameliorated glutathione and its dependent enzymes (glutathione peroxidase, glutathione reductase) which were depleted by Aβ(25-35) in PC 12 cells. These results were supported by the immunocytochemical finding that has shown protection of cells by TQ from noxious effects of Aβ(25-35). These results indicate that TQ holds potential for neuroprotection and may be a promising approach for the treatment of neurodegenerative disorders including AD.     

Resveratrol protects rats from Aβ-induced neurotoxicity by the reduction of iNOS expression and lipid peroxidation

Alzheimer disease (AD) is an age-dependent neurodegenerative disease characterized by the formation of β-amyloid (Aβ)-containing senile plaque. The disease could be induced by the administration of Aβ peptide, which was also known to upregulate inducible nitric oxide synthase (iNOS) and stimulate neuronal apoptosis. The present study is aimed to elucidate the cellular effect of resveratrol, a natural phytoestrogen with neuroprotective activities, on Aβ-induced hippocampal neuron loss and memory impairment. On adult Sprague-Dawley rats, we found the injection of Aβ could result in a significant impairment in spatial memory, a marked increase in the cellular level of iNOS and lipid peroxidation, and an apparent decrease in the expression of heme oxygenase-1 (HO-1). By combining the treatment with Aβ, resveratrol was able to confer a significant improvement in spatial memory, and protect animals from Aβ-induced neurotoxicity. These neurological protection effects of resveratrol were associated with a reduction in the cellular levels of iNOS and lipid peroxidation and an increase in the production of HO-1. Moreover, the similar neurological and cellular response were also observed when Aβ treatment was combined with the administration of a NOS inhibitor, N(G)-nitro-L-arginine methyl ester hydrochloride (L-NAME). These findings strongly implicate that iNOS is involved in the Aβ-induced lipid peroxidation and HO-1 downregulation, and resveratrol protects animals from Aβ-induced neurotoxicity by suppressing iNOS production.     

N-homocysteinylation of α-synuclein promotes its aggregation and neurotoxicity

The aggregation of α-synuclein plays a pivotal role in the pathogenesis of Parkinson's disease (PD). Epidemiological evidence indicates that high level of homocysteine (Hcy) is associated with an increased risk of PD. However, the molecular mechanisms remain elusive. Here, we report that homocysteine thiolactone (HTL), a reactive thioester of Hcy, covalently modifies α-synuclein on the K80 residue. The levels of α-synuclein K80Hcy in the brain are increased in an age-dependent manner in the TgA53T mice, correlating with elevated levels of Hcy and HTL in the brain during aging. The N-homocysteinylation of α-synuclein stimulates its aggregation and forms fibrils with enhanced seeding activity and neurotoxicity. Intrastriatal injection of homocysteinylated α-synuclein fibrils induces more severe α-synuclein pathology and motor deficits when compared with unmodified α-synuclein fibrils. Increasing the levels of Hcy aggravates α-synuclein neuropathology in a mouse model of PD. In contrast, blocking the N-homocysteinylation of α-synuclein ameliorates α-synuclein pathology and degeneration of dopaminergic neurons. These findings suggest that the covalent modification of α-synuclein by HTL promotes its aggregation. Targeting the N-homocysteinylation of α-synuclein could be a novel therapeutic strategy against PD.     

Extracellular chaperones prevent Aβ42-induced toxicity in rat brains

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterised by cognitive decline, formation of the extracellular amyloid β (Aβ₄₂) plaques, neuronal and synapse loss, and activated microglia and astrocytes. Extracellular chaperones, which are known to inhibit amyloid fibril formation and promote clearance of misfolded aggregates, have recently been shown to reduce efficiently the toxicity of HypF-N misfolded oligomers to immortalised cell lines, by binding and clustering them into large species. However, the role of extracellular chaperones on Aβ oligomer toxicity remains unclear, with reports often appearing contradictory. In this study we microinjected into the hippocampus of rat brains Aβ₄₂ oligomers pre-incubated for 1 h with two extracellular chaperones, namely clusterin and α₂-macroglobulin. The chaperones were found to prevent Aβ₄₂-induced learning and memory impairments, as assessed by the Morris Water Maze test, and reduce Aβ₄₂-induced glia inflammation and neuronal degeneration in rat brains, as probed by fluorescent immunohistochemical analyses. Moreover, the chaperones were able to prevent Aβ₄₂ colocalisation with PSD-95 at post-synaptic terminals of rat primary neurons, suppressing oligomer cytotoxicity. All such effects were not effective by adding pre-formed oligomers and chaperones without preincubation. Molecular chaperones have therefore the potential to prevent the early symptoms of AD, not just by inhibiting Aβ₄₂ aggregation, as previously demonstrated, but also by suppressing the toxicity of Aβ₄₂ oligomers after they are formed. These findings elect them as novel neuroprotectors against amyloid-induced injury and excellent candidates for the design of therapeutic strategies against AD.     

Inhibitory effect of curcumin on the Al(III)-induced Aβ₄₂ aggregation and neurotoxicity in vitro

The pathogenesis of Alzheimer's disease (AD) involves a key event which changes the morphology of amyloid-β 42 (Aβ)?? peptide from its soluble monomeric form into the fibrillated aggregates in the brain. Aluminum ion, Al(III), is known to act as a pathological chaperone of the Aβ?? in this process; curcumin, a natural phenolic compound, is considered capable of binding Al(III) and Aβ??; nevertheless, little is known about the combined action of curcumin and Al(III) on the Aβ?? fibrillation and neurotoxicity. Here, combinations of circular dichroism spectroscopy, thioflavin T fluorescence, atomic force microscopy, Bradford and MTT assays, it is demonstrated that although Al(III) can promote the Aβ?? fibrillation dose-dependently, leading to the high neurotoxicity to PC12 cells, curcumin can inhibit the events. Besides, we found that curcumin is able not only to inhibit the formation of Al(III)-induced Aβ?? fibrillation, but also to form the Al(III)-curcumin complexes which in turn can remold the preformed, mature, ordered Aβ?? fibrils into the low toxic amorphous aggregates. These findings suggest that curcumin could block the binding of Al(III) with Aβ?? and form the Al(III)-curcumin complexes, so as to inhibit the Al(III)-induced Aβ?? fibrillation and neurotoxicity. The Al(III)-curcumin complexes are worth potentially developing as a therapy agent against the neurodegenerative disorders in the future.     

Rosmarinic acid and ursolic acid alleviate deficits in cognition,synaptic regulation and adult hippocampal neurogenesis in an Aβ1-42-induced mouse model of Alzheimer's disease

BackgroundRosmarinus officinalis, commonly known as rosemary, is a medicinal herb that presents significant biological properties such as antimicrobial, antioxidant, anti-inflammatory, anti-diabetic and anti-depressant activities. Recent findings correlate impaired adult neurogenesis, which is crucial for the maintenance of synaptic plasticity and hippocampal functioning, synaptic regulation with the pathological hallmarks of Alzheimer's disease (AD). These observations call for the need to developing compounds that promote neurogenesis and alleviates deficits in cognition and synaptic regulation.Purpose and study designThe present study was conducted to determine the proneurogenic effects of R. officinalis and its active compounds (ursolic acid and rosmarinic acid) in comparison to Donepezil in an Aβ_1-42-induced mouse model of AD.MethodsBALB/c mice were divided into ten groups. Half were injected with Aβ_1-42 in the hippocampus through stereotaxic surgery to generate the disease groups. The other half received control injections. Each set of five groups were administered orally with vehicle, an ethanolic extract of R. officinalis, ursolic acid, rosmarinic acid or donepezil. Behavior analysis included the Morris water maze test, the novel object recognition test and the Elevated plus maze. The mice were then sacrificed and the hippocampal tissue was processed for immunohistochemistry and gene expression analysis.ResultsThe results show a protective effect by rosmarinic acid and ursolic acid in reversing the deficits in spatial and recognition memory as well as changes in anxiety induced by Aβ_1-42. The neuronal density and the expression levels of neurogenic (Ki67, NeuN and DCX) and synaptic (Syn I, II, III, Synaptophysin and PSD-95) markers were also normalized upon treatment with rosmarinic and ursolic acid.ConclusionOur findings indicate the potential of R. officinalis and its active compounds as therapeutic agents against Aβ_1-42-induced neurotoxicity in AD.     

Neuroprotective effects of overexpressed cyclophilin B against Aβ-induced neurotoxicity in PC12 cells

Accumulated amyloid-β (Aβ) is a well-known cause of neuronal apoptosis in Alzheimer disease and functions in part by generating oxidative stress. Our previous work suggested that cyclophilin B (CypB) protects against endoplasmic reticulum (ER) stress. Therefore, in this study we examined the ability of CypB to protect against Aβ toxicity. CypB is present in the neurons of rat and mouse brains, and treating neural cells with Aβ_25-35 mediates apoptotic cell death. Aβ_25-35-induced neuronal toxicity was inhibited by the overexpression of CypB as measured by cell viability, apoptotic morphology, sub-G1 cell population, intracellular reactive oxygen species accumulation, activated caspase-3, PARP cleavage, Bcl-2 proteins, mitogen-activated protein kinase (MAPK) activation, and phosphoinositide 3-kinase (PI-3-K) activation. CypB/R95A PPIase mutants did not reduce Aβ_25-35 toxicity. We showed that Aβ_25-35-induced apoptosis is more severe in a CypB knockdown model, confirming that CypB protects against Aβ_25-35-induced toxicity. Consequently, these findings suggest that CypB may protect against Aβ toxicity by its antioxidant properties, by regulating MAPK and PI-3-K signaling, and through the ER stress pathway.     

Brusatol inhibits amyloid-β-induced neurotoxicity in U-251 cells via regulating the Nrf2/HO-1 pathway

Amyloid-β (Aβ) has been reported to cause oxidative damage of neurons leading to neurotoxicity in a variety of diseases and cancers. As an anticancer drug, brusatol (BR) has been shown to have potent cytotoxic effects on various cancer cell lines. In this study, the effect and mechanism of BR on Aβ-induced neurotoxicity was investigated in U-251 glioma cells. Using the MTT assay, the results suggest that BR ameliorated cell injury induced by Aβ in U-251 cells. After running Hoechst and Western blot assays, BR prevented cell apoptosis induced by Aβ in U-251 cells. In addition, BR inhibited the increased reactive oxygen species and mitochondrial membrane potential levels induced by Aβ in U-251 cells using the DCFH-DA and Rh123 method. Furthermore, BR induced the Nrf2/HO-1 pathway by inhibiting the PI3K/AKT/mTOR pathway to inhibit neurotoxicity elicited by Aβ. These results suggest that brustasol is a valuable potential antitumor drug available for chemotherapy.     

The piperine derivative HJ105 inhibits Aβ1–42-induced neuroinflammation and oxidative damage via the Keap1-Nrf2-TXNIP axis

BackgroundPiperine is a great lead compound, as a phytopharmaceutical with reported neuroprotective effects in neurodegenerative diseases. HJ105, a piperine derivative with high affinity to Keap1 receptor, attracts increasing attention in Alzheimer's disease (AD) treatment.PurposeThis work mainly aimed to study HJ105’s therapeutic effects on Aβ_1–42-associated AD and the underpinning mechanisms.MethodsIn the in vivo part, a rat model of AD was established by bilateral intra-hippocampal administration of aggregated Aβ_1–42, followed by a month of intragastric HJ105 or donepezil administration. Spatial and learning memories were detected by the Morris water maze assay, passive avoidance learning as well as Y-maze test. The morphology of hippocampal neurons was assessed by hematoxylin-eosin (H&E) staining. In addition, the amounts of the IL-1β and TNF-α were obtained with specific ELISA kits. More importantly, apoptosis-related proteins and factors involved in Nrf2/TXNIP/NLPR3 pathways were detected by Western blot, while the interaction between Keap1 and Nrf2 was assessed by co-immunoprecipitation. In the in vitro part, human neuroblastoma (SH-SY5Y) cells were applied to evaluate the role of HJ105 on Aβ_1–42-induced neuronal damage.ResultsTreatment of HJ105 not only reversed memory impairment, but also protected neurons in the hippocampus by inhibiting Bax/Bcl2 ratio increase. HJ105 decreased TXNIP expression, suppressing NLRP3 inflammasome activation in the hippocampus, which in turn counteracted the upregulation of IL-1β and TNF-α. Notably, HJ105 exerted an inhibitory effect on Keap1-Nrf2 interaction and upregulated nuclear Nrf2, which conversely increased the expression levels of superoxide dismutase, catalase and glutathione peroxidase and downregulated malondialdehyde. Additionally, neurotoxicity induced by Aβ_1–42 in SH-SY5Y cells was alleviated by HJ105.ConclusionOverall, HJ105 exerts neuroprotective effects in SH-SY5Y cells induced by Aβ_1–42 as well as in experimental rats with AD by decreasing apoptosis, oxidative stress and neuroinflammation, partly via suppression of Keap1-Nrf2 complex generation. HJ105 might represent a promising compound for AD treatment.