Effect of total flavonoids of Dracocephalum moldavica L. On neuroinflammation in Alzheimer's disease model amyloid-ß (Aß1-42)-peptide-induced astrocyte activation.

One of the main pathological features noted in Alzheimer's disease (AD) is the presence of plagues of aggregated ß-amyloid (Aß1-42)-peptides. Excess deposition of amyloid-ß oligomers (AßO) are known to promote neuroinflammation. Sequentially, following neuroinflammation astrocytes become activated with cellular characteristics to initiate activated astrocytes. The purpose of this study was to determine whether total flavonoids derived from Dracocephalum moldavica L. (TFDM) inhibited Aß1-42-induced damage attributed to activated C8-D1A astrocytes. Western blotting and ELISA were used to determine the expression of glial fibrillary acidic protein (GFAP), and complement C3 to establish the activation status of astrocytes following induction from exposure to Aß1-42. Data demonstrated that stimulation of C8-D1A astrocytes by treatment with 40 µM Aß1-42 for 24 hr produced significant elevation in protein expression and protein levels of acidic protein (GFAP) and complement C3 accompanied by increased expression and levels of inflammatory cytokines. Treatment with TFDM or the clinically employed drug donepezil in AD therapy reduced production of inflammatory cytokines, and toxicity initiated following activation of C8-D1A astrocytes following exposure to Aß1-42. Therefore, TFDM similar to donepezil inhibited inflammatory secretion in reactive astrocytes, suggesting that TFDM may be considered as a potential compound to be utilized in AD therapy.

Blockage of metallothionein synthesis via adrenaline ß receptor activation invalidates dehydroeffusol-mediated prevention of amyloid ß1-42 toxicity.

Dehydroeffusol, a major phenanthrene in Juncus effusus, protects neurodegeneration induced by intracellular Zn2+ ferried by extracellular amyloid ß1-42 (Aß1-42). Here we focused on adrenaline ß receptor activation and the induction of metallothioneins (MTs), intracellular Zn2+-binding proteins to test the protective mechanism of dehydroeffusol. Isoproterenol, an agonist of adrenergic ß receptors elevated the level of MTs in the dentate granule cell layer 1 day after intracerebroventricular (ICV) injection. When Aß1-42 was injected 1 day after isoproterenol injection, pre-injection of isoproterenol protected Aß1-42 toxicity via reducing the increase in intracellular Zn2+ after ICV injection of Aß1-42. On the basis of the effect of increased MTs by isoproterenol, dehydroeffusol (15 mg/kg body weight) was orally administered to mice once a day for 2 days. On day later, dehydroeffusol elevated the level of MTs and prevented Aß1-42 toxicity via reducing Aß1-42-mediated increase in intracellular Zn2+. In contrast, propranolol, an antagonist of adrenergic ß receptors reduced the level of MTs increased by dehydroeffusol, resulting in invalidating the preventive effect of dehydroeffusol on Aß1-42 toxicity. The present study indicates that blockage of MT synthesis via adrenaline ß receptor activation invalidates dehydroeffusol-mediated prevention of Aß1-42 toxicity. It is likely that MT synthesis via adrenaline ß receptor activation is beneficial to neuroprotection and that oral intake of dehydroeffusol preventively serves against the Aß1-42 toxicity.

A Chronic Increase in Blood-Brain Barrier Permeability Facilitates Intraneuronal Deposition of Exogenous Bloodborne Amyloid-Beta1-42 Peptide in the Brain and Leads to Alzheimer's Disease-Relevant Cognitive Changes in a Mouse Model.

Background: Increased blood-brain barrier (BBB) permeability and amyloid-ß (Aß) peptides (especially Aß1-42) (Aß42) have been linked to Alzheimer's disease (AD) pathogenesis, but the nature of their involvement in AD-related neuropathological changes leading to cognitive changes remains poorly understood. Objective: To test the hypothesis that chronic extravasation of bloodborne Aß42 peptide and brain-reactive autoantibodies and their entry into the brain parenchyma via a permeable BBB contribute to AD-related pathological changes and cognitive changes in a mouse model. Methods: The BBB was rendered chronically permeable through repeated injections of Pertussis toxin (PT), and soluble monomeric, fluorescein isothiocyanate (FITC)-labeled or unlabeled Aß42 was injected into the tail-vein of 10-month-old male CD1 mice at designated intervals spanning ∼3 months. Acquisition of learned behaviors and long-term retention were assessed via a battery of cognitive and behavioral tests and linked to neuropathological changes. Results: Mice injected with both PT and Aß42 demonstrated a preferential deficit in the capacity for long-term retention and an increased susceptibility to interference in selective attention compared to mice exposed to PT or saline only. Immunohistochemical analyses revealed increased BBB permeability and entry of bloodborne Aß42 and immunoglobulin G (IgG) into the brain parenchyma, selective neuronal binding of IgG and neuronal accumulation of Aß42 in animals injected with both PT and Aß42 compared to controls. Conclusion: Results highlight the potential synergistic role of BBB compromise and the influx of bloodborne Aß42 into the brain in both the initiation and progression of neuropathologic and cognitive changes associated with AD.

Impact of nanoplastics on Alzheimer 's disease: Enhanced amyloid-ß peptide aggregation and augmented neurotoxicity.

Nanoplastics, widely existing in the environment and organisms, have been proven to cross the blood-brain barrier, increasing the incidence of neurodegenerative diseases like Alzheimer's disease (AD). However, current studies mainly focus on the neurotoxicity of nanoplastics themselves, neglecting their synergistic effects with other biomolecules and the resulting neurotoxicity. Amyloid ß peptide (Aß), which triggers neurotoxicity through its self-aggregation, is the paramount pathogenic protein in AD. Here, employing polystyrene nanoparticles (PS) as a model for nanoplastics, we reveal that 100 pM PS nanoparticles significantly accelerate the nucleation rate of two Aß subtypes (Aß40 and Aß42) at low concentrations, promoting the formation of more Aß oligomers and leading to evident neurotoxicity. The hydrophobic surface of PS facilitates the interaction of hydrophobic fragments between Aß monomers, responsible for the augmented neurotoxicity. This work provides consequential insights into the modulatory impact of low-dose PS on Aß aggregation and the ensuing neurotoxicity, presenting a valuable foundation for future research on the intricate interplay between environmental toxins and brain diseases.

A single-domain antibody detects and neutralises toxic Aß42 oligomers in the Alzheimer's disease CSF.

BACKGROUND: Amyloid-ß42 (Aß42) aggregation consists of a complex chain of nucleation events producing soluble oligomeric intermediates, which are considered the major neurotoxic agents in Alzheimer's disease (AD). Cerebral lesions in the brain of AD patients start to develop 20 years before symptom onset; however, no preventive strategies, effective treatments, or specific and sensitive diagnostic tests to identify people with early-stage AD are currently available. In addition, the isolation and characterisation of neurotoxic Aß42 oligomers are particularly difficult because of their transient and heterogeneous nature. To overcome this challenge, a rationally designed method generated a single-domain antibody (sdAb), named DesAb-O, targeting Aß42 oligomers. METHODS: We investigated the ability of DesAb-O to selectively detect preformed Aß42 oligomers both in vitro and in cultured neuronal cells, by using dot-blot, ELISA immunoassay and super-resolution STED microscopy, and to counteract the toxicity induced by the oligomers, monitoring their interaction with neuronal membrane and the resulting mitochondrial impairment. We then applied this approach to CSF samples (CSFs) from AD patients as compared to age-matched control subjects. RESULTS: DesAb-O was found to selectively detect synthetic Aß42 oligomers both in vitro and in cultured cells, and to neutralise their associated neuronal dysfunction. DesAb-O can also identify Aß42 oligomers present in the CSFs of AD patients with respect to healthy individuals, and completely prevent cell dysfunction induced by the administration of CSFs to neuronal cells. CONCLUSIONS: Taken together, our data indicate a promising method for the improvement of an early diagnosis of AD and for the generation of novel therapeutic approaches based on sdAbs for the treatment of AD and other devastating neurodegenerative conditions.

Neuroprotective Effects of Ferrostatin and Necrostatin Against Entorhinal Amyloidopathy-Induced Electrophysiological Alterations Mediated by voltage-gated Ca2+ Channels in the Dentate Gyrus Granular Cells.

Alzheimer's disease (AD) is a progressive neurodegenerative disease that is the main form of dementia. Abnormal deposition of amyloid-beta (Aß) peptides in neurons and synapses cause neuronal loss and cognitive deficits. We have previously reported that ferroptosis and necroptosis were implicated in Aß25-35 neurotoxicity, and their specific inhibitors had attenuating effects on cognitive impairment induced by Aß25-35 neurotoxicity. Here, we aimed to examine the impact of ferroptosis and necroptosis inhibition following the Aß25-35 neurotoxicity on the neuronal excitability of dentate gyrus (DG) and the possible involvement of voltage-gated Ca2+ channels in their effects. After inducing Aß25-35 neurotoxicity, electrophysiological alterations in the intrinsic properties and excitability were recorded by the whole-cell patch-clamp under current-clamp condition. Voltage-clamp recordings were also performed to shed light on the involvement of calcium channel currents. Aß25-35 neurotoxicity induced a considerable reduction in input resistance (Rin), accompanied by a profoundly decreased excitability and a reduction in the amplitude of voltage-gated calcium channel currents in the DG granule cells. However, three days of administration of either ferrostatin-1 (Fer-1), a ferroptosis inhibitor, or Necrostatin-1 (Nec-1), a necroptosis inhibitor, in the entorhinal cortex could almost preserve the normal excitability and the Ca2+ currents. In conclusion, these findings suggest that ferroptosis and necroptosis involvement in EC amyloidopathy could be a potential candidate to prevent the suppressive effect of Aß on the Ca2+ channel current and neuronal function, which might take place in neurons during the development of AD.

GDP dissociation inhibitor 1 (GDI1) attenuates ß-amyloid-induced neurotoxicity in Alzheimer's diseases.

OBJECTIVE: ß-Amyloid (Aß) induced neurotoxicity is an implicated mechanism in Alzheimer's disease (AD). This study focused on the role of GDP dissociation inhibitor 1 (GDI1) in Aß-induced neurotoxicity. METHODS: Data from the GEO database for AD-related datasets GSE140829, GSE63061, GSE36980, and GSE60360 were downloaded and identified common differentially expressed genes (coDEGs). The mRNA levels of GDI1 in the serum of AD patients were analyzed by RT-qPCR. ROC curve evaluated the diagnostic value. Aß25-35 induced SH-SY5Y cells to construct an AD cell model. CCK-8, flow cytometry, and ELISA assay were used to analyze cell viability, apoptosis, and concentrations of inflammatory factors. KEGG enrichment was employed to analyze the signal pathway of targets from GDI1 in the AD. RESULTS: The GEO database identifies coDEGs including GDI1. GDI1 is generally elevated in serum from AD patients as well as in Aß-induced SH-SY5Y cells. GDI1 has 77.97% sensitivity and 84.44% specificity to identify AD patients from controls. Aß induced decreased cell viability, increased apoptosis, and promoted over-secretion of inflammatory cytokines, but they were all partially weakened by reduced GDI1. What's more, the GDI1 interacting gene and AD target gene were co-enriched on Endocytosis and MAPK signaling pathway. CONCLUSION: Elevated GDI1 is a potential diagnostic biomarker for AD and that inhibition of GDI1 attenuates Aß-induced neurotoxicity in AD. Our study offers new horizons for AD treatment.

Hesperidin Methyl Chalcone reduces extracellular Aß(25-35) peptide aggregation and fibrillation and also protects Neuro 2a cells from Aß(25-35) induced neuronal dysfunction.

In the present study, we evaluated the neuroprotective potential of Hesperidin Methyl Chalcone (HMC) against the neurotoxicity induced by Aß(25-35) peptide. HMC demonstrated higher free-radical scavenging activity than Hesperidin in initial cell-free studies. Investigations using the fluorescent dye thioflavin T with Aß(25-35) peptide showed that HMC has the ability to combat extracellular amyloid aggregation by possessing anti-aggregation property against oligomers and by disaggregating mature fibrils. Also, the results of the molecular simulation studies show that HMC ameliorated oligomer formation. Further, the anti-Alzheimer's property of HMC was investigated in in vitro cell conditions by pre-treating the neuro 2a (N2a) cells with HMC before inducing Aß(25-35) toxicity. The findings demonstrate that HMC increased cell viability, reduced oxidative stress, prevented macromolecular damage, allayed mitochondrial dysfunction, and exhibited anticholinesterase activity. HMC also reduced Aß induced neuronal cell death by modulating caspase-3 activity, Bax expression and Bcl2 overexpression, demonstrating that HMC pre-treatment reduced mitochondrial damage and intrinsic apoptosis induced by Aß(25-35).In silico evaluation against potential AD targets reveal that HMC could be a potent inhibitor of BACE-1, inhibiting the formation of toxic Aß peptides. Overall, the findings imply that the neuroprotective efficacy of HMC has high prospects for addressing a variety of pathogenic consequences caused by amyloid beta in AD situations and alleviating cognitive impairments.

The 3-(3-oxoisoindolin-1-yl)pentane-2,4-dione (ISOAC1) as a new molecule able to inhibit Amyloid ß aggregation and neurotoxicity.

Amyloid ß 1-42 (Aß1-42) protein aggregation is considered one of the main triggers of Alzheimer's disease (AD). In this study, we examined the in vitro anti-amyloidogenic activity of the isoindolinone derivative 3-(3-oxoisoindolin-1-yl)pentane-2,4-dione (ISOAC1) and its neuroprotective potential against the Aß1-42 toxicity. By performing the Thioflavin T fluorescence assay, Western blotting analyses, and Circular Dichroism experiments, we found that ISOAC1 was able to reduce the Aß1-42 aggregation and conformational transition towards ß-sheet structures. Interestingly, in silico studies revealed that ISOAC1 was able to bind to both the monomer and a pentameric protofibril of Aß1-42, establishing a hydrophobic interaction with the PHE19 residue of the Aß1-42 KLVFF motif. In vitro analyses on primary cortical neurons showed that ISOAC1 counteracted the increase of intracellular Ca2+ levels and decreased the Aß1-42-induced toxicity, in terms of mitochondrial activity reduction and increase of reactive oxygen species production. In addition, confocal microscopy analyses showed that ISOAC1 was able to reduce the Aß1-42 intraneuronal accumulation. Collectively, our results clearly show that ISOAC1 exerts a neuroprotective effect by reducing the Aß1-42 aggregation and toxicity, hence emerging as a promising compound for the development of new Aß-targeting therapeutic strategies for AD treatment.

Hederagenin Upregulates PTPN1 Expression in Aß-Stimulated Neuronal Cells, Exerting Anti-Oxidative Stress and Anti-Apoptotic Activities.

Alzheimer's disease (AD) is a prevalently neurodegenerative disease characterized by neuronal damage which is associated with amyloid-ß (Aß) accumulation. Hederagenin is a triterpenoid saponin, exerting anti-apoptotic, anti-oxidative, anti-inflammatory, anti-tumoral, and neuroprotective activities. However, its role in AD progression is still obscure. The aim of this study was to explore the influences of hederagenin on Aß-caused neuronal injury in vitro. Neuronal cells were treated with Aß25-35 (Aß) to establish a cellular model of AD. Cell viability was assessed using cell counting kit-8 (CCK-8). Oxidative stress was evaluated by detecting reactive oxygen species (ROS) generation and superoxide dismutase (SOD) activity. Apoptosis was investigated using TUNEL staining and caspase-3 activity assays. Protein tyrosine phosphatase nonreceptor type 1 (PTPN1) was screened by bioinformatics analysis. Protein levels of PTPN1 and protein kinase B (Akt) were measured by western blotting. Hederagenin (2.5, 5, and 10 µM) alone did not affect viability of neuronal cells, but relieved Aß-induced viability reduction. Hederagenin mitigated Aß-induced increase in ROS accumulation and decrease in SOD activity. Hederagenin attenuated Aß-induced increase in apoptotic rate and caspase-3 activity. PTPN1 was screened as a target of hederagenin against AD by bioinformatics analysis. Hederagenin treatment resisted Aß-induced decrease in PTPN1 mRNA and protein levels in neuronal cells. PTPN1 silencing attenuated the suppressive functions of hederagenin in Aß-stimulated oxidative stress and apoptosis. Hederagenin mitigated Aß-induced Akt signaling inactivation by upregulating PTPN1 expression. In conclusion, hederagenin attenuates oxidative stress and apoptosis in neuronal cells stimulated with Aß by promoting PTPN1/Akt signaling activation.

Beta Amyloid Oligomers with Higher Cytotoxicity have Higher Sidechain Dynamics.

The underlying biophysical principle governing the cytotoxicity of the oligomeric aggregates of ß-amyloid (Aß) peptides has long been an enigma. Here we show that the size of Aß40 oligomers can be actively controlled by incubating the peptides in reverse micelles. Our approach allowed for the first time a detailed comparison of the structures and dynamics of two Aß40 oligomers of different sizes, viz., 10 and 23 nm, by solid-state NMR. From the chemical shift data, we infer that the conformation and/or the chemical environments of the residues from K16 to K28 are different between the 10-nm and 23-nm oligomers. We find that the 10-nm oligomers are more cytotoxic, and the molecular motion of the sidechain of its charged residue K16 is more dynamic. Interestingly, the residue A21 exhibits unusually high structural rigidity. Our data raise an interesting possibility that the cytotoxicity of Aß40 oligomers could also be correlated to the motional dynamics of the sidechains.

Towards the Integrative Theory of Alzheimer's Disease: Linking Molecular Mechanisms of Neurotoxicity, Beta-amyloid Biomarkers, and the Diagnosis.

INTRODUCTION: A major gap in amyloid-centric theories of Alzheimer's disease (AD) is that even though amyloid fibrils per se are not toxic in vitro, the diagnosis of AD clearly correlates with the density of beta-amyloid (Aß) deposits. Based on our proposed amyloid degradation toxicity hypothesis, we developed a mathematical model explaining this discrepancy. It suggests that cytotoxicity depends on the cellular uptake of soluble Aß rather than on the presence of amyloid aggregates. The dynamics of soluble beta-amyloid in the cerebrospinal fluid (CSF) and the density of Aß deposits is described using a system of differential equations. In the model, cytotoxic damage is proportional to the cellular uptake of Aß, while the probability of an AD diagnosis is defined by the Aß cytotoxicity accumulated over the duration of the disease. After uptake, Aß is concentrated intralysosomally, promoting the formation of fibrillation seeds inside cells. These seeds cannot be digested and are either accumulated intracellularly or exocytosed. Aß starts aggregating on the extracellular seeds and, therefore, decreases in concentration in the interstitial fluid. The dependence of both Aß toxicity and aggregation on the same process-cellular uptake of Aß-explains the correlation between AD diagnosis and the density of amyloid aggregates in the brain. METHODS: We tested the model using clinical data obtained from the Alzheimer's Disease Neuroimaging Initiative (ADNI), which included records of beta-amyloid concentration in the cerebrospinal fluid (CSF-Aß42) and the density of beta-amyloid deposits measured using positron emission tomography (PET). The model predicts the probability of AD diagnosis as a function of CSF-Aß42 and PET and fits the experimental data at the 95% confidence level. RESULTS: Our study shows that existing clinical data allows for the inference of kinetic parameters describing beta-amyloid turnover and disease progression. Each combination of CSF-Aß42 and PET values can be used to calculate the individual's cellular uptake rate, the effective disease duration, and the accumulated toxicity. We show that natural limitations on these parameters explain the characteristic distribution of the clinical dataset for these two biomarkers in the population. CONCLUSION: The resulting mathematical model interprets the positive correlation between the density of Aß deposits and the probability of an AD diagnosis without assuming any cytotoxicity of the aggregated beta-amyloid. To the best of our knowledge, this model is the first to mechanistically explain the negative correlation between the concentration of Aß42 in the CSF and the probability of an AD diagnosis. Finally, based on the amyloid degradation toxicity hypothesis and the insights provided by mathematical modeling, we propose new pathophysiology-relevant biomarkers to diagnose and predict AD.

The Role of Protein-L-isoaspartyl Methyltransferase (PIMT) in the Suppression of Toxicity of the Oligomeric Form of Aß42, in Addition to the Inhibition of Its Fibrillization.

The oligomeric form of amyloid-ß peptide (Aß42) plays a crucial role in the pathogenesis of Alzheimer's disease (AD) and is responsible for cognitive deficits. The soluble oligomers are believed to be more toxic compared to the fibril form. Protein-L-isoaspartyl methyltransferase (PIMT) is a repair enzyme that converts aberrant isoAsp residues, formed spontaneously on isomerization of normal Asp and Asn residues, back to typical Asp. It was shown to inhibit the fibrillization of Aß42 (containing three Asp residues), and here, we investigate its effect on the size, conformation, and toxicity of Aß42 oligomers (AßO). Far-UV CD indicated a shift in the conformational feature of AßOs from the random coil to ß-sheet in the presence of PIMT. Binding of bis-ANS to different AßOs (obtained using different concentrations of Aß42 monomer) indicated the correlation of size of oligomers to hydrophobicity: the smallest AßO having the highest hydrophobicity is the most toxic. Dynamic light scattering showed an increase in size of AßO with the addition of PIMT, a contrasting role to that on Aß fibril. Assays using PC12-derived neurons showed the neuroprotective role of PIMT against AßO-induced toxicity. Furthermore, we have elaborated on the molecular mechanism of the antifibrillar action of PIMT and how this function is correlated with its enzymatic activity. PIMT has a more pronounced effect on AßO as compared to a small heat shock protein, pointing to its importance for the amelioration of the adverse effect of both Aß42 oligomers and fibrils.

Assessing the Safety of MA-[D-Leu-4]-OB3, a Synthetic Peptide Leptin Mimetic: Two Pre-Clinical Toxicity Studies in Male and Female C57BL/6 Mice.

Although the regulatory influence of leptin on energy balance, glycemic control, immunity, reproduction, and cognition is well established, its clinical application to common obesity and its co-morbidities has been limited by impaired transport across the blood-brain barrier, and tendencies to induce adverse side effects. To circumvent these drawbacks, MA-[D-Leu-4]-OB3, a leptin-related synthetic peptide that mimics the metabolic and neurotrophic effects of leptin in mouse models of genetic and non-genetic obesity, diabetes, and cognitive dysfunction, has been developed. This report presents the results of our initial efforts to assess the safety of orally delivered MA-[D-Leu-4]-OB3. Two pre-clinical studies were done in male and female C57BL/6 mice: a short-term study with a high dose of MA-[D-Leu-4]-OB3 (50 mg/kg/100 µL/day) and a dose-response study with 3 increasing concentrations of MA-[D-Leu-4]-OB3 (16.6, 50, and 150 mg/kg/100 µL/day). Body weight, food and water intake, glucose tolerance, and episodic memory were evaluated. Once-daily cage-side clinical observations were conducted to detect any physical or behavioral indicators of toxicity. Our results indicate that all metabolic and neurologic endpoints tested were either unaffected or improved by MA-[D-Leu-4]-OB3, and no clinical indicators of toxicity were evident. Together with our previously reported efficacy data, these results provide additional evidence supporting further development of this novel synthetic peptide leptin mimetic as a first-in-class peptide drug candidate for the treatment of a number of metabolic and/or cognitive dysfunctions in humans.

Oxymatrine-mediated prevention of amyloid ß-peptide-induced apoptosis on Alzheimer's model PC12 cells: in vitro cell culture studies and in vivo cognitive assessment in rats.

Alzheimer's disease (AD) is a major neurological disease affecting elderly individuals worldwide. Existing drugs only reduce the symptoms of the disease without addressing the underlying causes. Commonly, Aß25-35 peptide aggregation is the main reason for AD development. Recently, the discovery of multiple protein-targeting molecules has provided a new strategy for treating AD. This study demonstrates the neuroprotective potential of oxymatrine against multiple mechanisms, such as acetylcholinesterase, mitochondrial damage, and ß-amyloid-induced cell toxicity. The in vitro cell culture studies showed that oxymatrine possesses significant potential to inhibit acetylcholine esterase and promotes antioxidant, antiapoptotic effects while preventing Aß25-35 peptide aggregation in PC12 cells. Furthermore, oxymatrine protects PC12 cells against Aß25-35-induced cytotoxicity and down-regulates the reactive oxygen species generation. The in vivo acute toxicological studies confirm the safety of oxymatrine without causing organ damage or death in animals. Overall, this study provided evidence that oxymatrine is an efficient neuroprotective agent, with a potential to be a multifunctional drug for Alzheimer's disease treatment. These findings present a reliable and synergistic approach for treating AD.

Attenuation Aß1-42-induced neurotoxicity in neuronal cell by 660nm and 810nm LED light irradiation.

Oligomeric amyloid-ß 1-42 (Aß1-42) has a close correlation with neurodegenerative disorder especially Alzheimer's disease (AD). It induces oxidative stress and mitochondrial damage in neurons. Therefore, it is used to generate AD-like in vitro model for studying neurotoxicity and neuroprotection against amyloid-ß. A low-level light therapy (LLLT) is a non-invasive method that has been used to treat several neurodegenerative disorders. In this study, the red wavelength (660nm) and near infrared wavelength (810nm) at energy densities of 1, 3, and 5 J/cm2 were used to modulate biochemical processes in the neural cells. The exposure of Aß1-42 resulted in cell death, increased intracellular reactive oxygen species (ROS), and retracted neurite outgrowth. We showed that both of LLLT wavelengths could protect neurons form Aß1-42-induced neurotoxicity in a biphasic manner. The treatment of LLLT at 3 J/cm2 potentially alleviated cell death and recovered neurite outgrowth. In addition, the treatment of LLLT following Aß1-42 exposure could attenuate the intracellular ROS generation and Ca2+ influx. Interestingly, both wavelengths could induce minimal level of ROS generation. However, they did not affect cell viability. In addition, LLLT also stimulated Ca2+ influx, but not altered mitochondrial membrane potential. This finding indicated LLLT may protect neurons through the stimulation of secondary signaling messengers such as ROS and Ca2+. The increase of these secondary messengers was in a functional level and did not harmful to the cells. These results suggested the use of LLLT as a tool to modulate the neuronal toxicity following Aß1-42 accumulation in AD's brain.

Inhibition of Amyloid ß Aggregation and Cytotoxicity by Berbamine Hydrochloride.

Alzheimer's disease (AD) continues to be a major global health challenge, and the recent approval of Aduhelm and Leqembi has opened new avenues for its treatment. Small-molecule inhibitors targeting Aß aggregation hold promise as an alternative to monoclonal antibodies. In this study, we evaluated the ability of berbamine hydrochloride (BBMH), a member of the bisbenzylisoquinoline alkaloids, to reduce Aß aggregation and cytotoxicity. Thioflavin T kinetics, circular dichroism spectroscopy, and atomic force microscopy results indicated that BBMH effectively inhibited Aß aggregation. Surface plasmon resonance and molecular docking results further revealed that BBMH could bind to Aß fibrils, thereby hindering the aggregation process. This physical picture has been confirmed in a quantitative way by chemical kinetics analysis, which showed BBMH tends to bind with the fibril ends and thus prevents the transition from protofibrils to mature fibrils as well as the elongation process. Additionally, our MTT results showed that BBMH was able to reduce the cytotoxicity of Aß40 on N2a cells. Our results demonstrate, for the first time, the potential of BBMH to inhibit Aß aggregation and cytotoxicity, offering a promising direction for further research and drug development efforts in the fight against Alzheimer's disease.

Kidney Yang Deficiency Syndrome Exacerbates Aß25-35-Induced Pathological Changes, and Ginsenoside Re Ameliorates Synapse Lesions in Aß25-35- Injected Rats with Kidney Yang Deficiency Syndrome.

BACKGROUND: Traditional Chinese medicine (TCM) indicates that Alzheimer's disease (AD) is considered the consequence produced by Kidney Yang Deficiency Syndrome (KDS-Yang), which has similar clinical characteristics to glucocorticoid withdrawal syndrome. Ginsenoside Re (G-Re) has been found to ameliorate the symptoms and pathological impairments of AD. However, it's not clear whether G-Re could protect memory and synapse lesions against kidney deficiency dementia. METHODS: Subcutaneous injection of hydrocortisone for 14 days was used to produce KDS-Yang. On the 15th day, Aß25-35 peptide was injected into the intracerebroventricular (icv) of KDS-Yang rats. Spine density was analyzed by Golgi staining and the ultrastructural morphology of the synapse was detected using Transmission Electron Microscopy (TEM). Western blot was used to examine the expression of pS396, pS404, Tau-5, tGSK-3ß, pS9GSK-3ß, Syt, Syn I, GluA1, GluN2B, PSD93, PSD95, ß2-AR and pS346-b2-AR. RESULTS: Hyperphosphorylation of tau in Aß25-35-injected rats with KDS-Yang was stronger than in Aß25-35-injected rats at the sites of Ser396 and Ser404. G-Re improved spatial memory damage detected by Morris water-maze (MWM), enhanced spines density, the thickness of postsynaptic density (PSD) and increased the expression of Syt, Syn I, GluA1, GluN2B, PSD93 and PSD95. Moreover, GRe decreased the hyperphosphorylation of ß2-AR at serine 346 in Aß25-35-injected rats with KDS-Yang. CONCLUSION: KDS-Yang might exacerbate AD pathological lesions. Importantly, G-Re is a potential ingredient for protecting against memory and synapse deficits in kidney deficiency dementia.

Five-mer peptides prevent short-term spatial memory deficits in Aß25-35-induced Alzheimer's model mouse by suppressing Aß25-35 aggregation and resolving its aggregate form.

BACKGROUND: The development of drugs for Alzheimer's disease (AD), which is related to the misfolding and aggregation of amyloid-ß (Aß), is high in demand due to the growing number of AD patients. In this study, we screened 22 kinds of 5-mer synthetic peptides derived from the Box A region of Tob1 protein to find a peptide effective against Aß aggregation. METHODS: A Thioflavin T (ThT) assay was performed to evaluate aggregation and screen aggregation inhibitors. Male ICR mice (6 weeks old) were administered saline, 9 nmol Aß25-35, or a mixture of 9 nmol Aß25-35 and 9 nmol GSGFK in the right lateral ventricle. Short-term spatial memory was assessed through Y-maze. Microglia cells (BV-)2 cells were plated on 24-well plates (4 × 104 cells/well) and incubated for 48 h, and then, the cells were treated with 0.01, 0.05, 0.1, 0.2, or 0.5 mM GSGFK. After incubation for 24 h, bead uptake was evaluated using a laser confocal microscope and Cytation 5. RESULTS: We found two kinds of peptides, GSGNR and GSGFK, that were not only suppressed by aggregation of Aß25-35 but also resolved the aggregated Aß25-35. Results obtained from the Y-maze test on an Aß25-35-induced AD model mouse indicated that GSGFK prevents the deficits in short-term memory induced by Aß25-35. The effect of GSGFK on phagocytosis in BV-2 cells proved that GSGFK activates the phagocytic ability of microglia. CONCLUSIONS: In conclusion, 5-mer peptides prevent short-term memory deficit in Aß25-35 induced AD model mouse by reducing the aggregated Aß25-35. They may also upregulate the phagocytic ability of microglia, which makes 5-mer peptides suitable candidates as therapeutic drugs against AD.

Investigation of Cyclo-Z Therapeutic Effect on Insulin Pathway in Alzheimer's Rat Model: Biochemical and Electrophysiological Parameters.

Cyclo (his-pro-CHP) plus zinc (Zn+2) (Cyclo-Z) is the only known chemical that increases the production of insulin-degrading enzyme (IDE) and decreases the number of inactive insulin fragments in cells. The aim of the present study was to systematically characterize the effects of Cyclo-Z on the insulin pathway, memory functions, and brain oscillations in the Alzheimer's disease (AD) rat model. The rat model of AD was established by bilateral injection of Aß42 oligomer (2,5nmol/10µl) into the lateral ventricles. Cyclo-Z (10mg Zn+2/kg and 0.2mg CHP/kg) gavage treatment started seven days after Aß injection and lasted for 21 days. At the end of the experimental period, memory tests and electrophysiological recordings were performed, which were followed by the biochemical analysis. Aß42 oligomers led to a significant increase in fasting blood glucose, serum insulin, Homeostatic Model Assessment for Insulin Resistance (HOMA-IR) and phospho-tau-Ser356 levels. Moreover, Aß42 oligomers caused a significant decrement in body weight, hippocampal insulin, brain insulin receptor substrate (IRS-Ser612), and glycogen synthase kinase-3 beta (GSK-3ß) levels. Also, Aß42 oligomers resulted in a significant reduction in memory. The Cyclo-Z treatment prevented the observed alterations in the ADZ group except for phospho-tau levels and attenuated the increased Aß42 oligomer levels in the ADZ group. We also found that the Aß42 oligomer decreased the left temporal spindle and delta power during ketamine anesthesia. Cyclo-Z treatment reversed the Aß42 oligomer-related alterations in the left temporal spindle power. Cyclo-Z prevents Aß oligomer-induced changes in the insulin pathway and amyloid toxicity, and may contribute to the improvement of memory deficits and neural network dynamics in this rat model.