Alzheimer's Disease Cellular Model Research Articles

Combination of Secondary Plant Metabolites and Micronutrients Improves Mitochondrial Function in a Cell Model of Early Alzheimer's Disease.

Alzheimer's disease (AD) is characterized by excessive formation of beta-amyloid peptides (Aβ), mitochondrial dysfunction, enhanced production of reactive oxygen species (ROS), and altered glycolysis. Since the disease is currently not curable, preventive and supportive approaches are in the focus of science. Based on studies of promising single substances, the present study used a mixture (cocktail, SC) of compounds consisting of hesperetin (HstP), magnesium-orotate (MgOr), and folic acid (Fol), as well as the combination (KCC) of caffeine (Cof), kahweol (KW) and cafestol (CF). For all compounds, we showed positive results in SH-SY5Y-APP695 cells-a model of early AD. Thus, SH-SY5Y-APP695 cells were incubated with SC and the activity of the mitochondrial respiration chain complexes were measured, as well as levels of ATP, Aβ, ROS, lactate and pyruvate. Incubation of SH-SY5Y-APP695 cells with SC significantly increased the endogenous respiration of mitochondria and ATP levels, while Aβ1-40 levels were significantly decreased. Incubation with SC showed no significant effects on oxidative stress and glycolysis. In summary, this combination of compounds with proven effects on mitochondrial parameters has the potential to improve mitochondrial dysfunction in a cellular model of AD.

MicroRNA-128 suppresses tau phosphorylation and reduces amyloid-beta accumulation by inhibiting the expression of GSK3β, APPBP2, and mTOR in Alzheimer's disease.

Alzheimer's disease (AD) is characterized by the abnormal accumulation of hyperphosphorylated tau proteins and amyloid-beta (Aβ) peptides. Recent studies have shown that many microRNAs (miRNAs) are dysregulated in AD, and modulation of these miRNAs can influence the development of tau and Aβ pathology. The brain-specific miRNA miR-128, encoded by MIR128-1 and MIR128-2, is important for brain development and dysregulated in AD. In this study, the role of miR-128 in tau and Aβ pathology as well as the regulatory mechanism underlying its dysregulation were investigated. The effect of miR-128 on tau phosphorylation and Aβ accumulation was examined in AD cellular models through miR-128 overexpression and inhibition. The therapeutic potential of miR-128 in AD mouse model was assessed by comparing phenotypes of 5XFAD mice administered with miR-128-expressing AAVs with 5XFAD mice administered with control AAVs. Phenotypes examined included behavior, plaque load, and protein expression. The regulatory factor of miR-128 transcription was identified through luciferase reporter assay and validated by siRNA knockdown and ChIP analysis. Both gain-of-function and loss-of-function studies in AD cellular models reveal that miR-128 represses tau phosphorylation and Aβ secretion. Subsequent investigations show that miR-128 directly inhibits the expression of tau phosphorylation kinase GSK3β and Aβ modulators APPBP2 and mTOR. Upregulation of miR-128 in the hippocampus of 5XFAD mice ameliorates learning and memory impairments, decreases plaque deposition, and enhances autophagic flux. We further demonstrated that C/EBPα transactivates MIR128-1 transcription, while both C/EBPα and miR-128 expression are inhibited by Aβ. Our findings suggest that miR-128 suppresses AD pathogenesis, and could be a promising therapeutic target for AD. We also find a possible mechanism underlying the dysregulation of miR-128 in AD, in which Aβ reduces miR-128 expression by inhibiting C/EBPα.

N-methyl-D-aspartic acid receptor 2A functionalized stationary phase: A reliable method for pursuing potential ligands against Alzheimer's disease from natural products.

N-methyl-D-aspartic acid (NMDA) receptors play subunit-specific role in central neuronal development. However, insights into the pharmacological modulation of NMDA receptors were mainly lack of subunit and synaptic selectivity. The purpose of the present study was to develop a novel strategy to rapidly recognize NMDA subunit 2A (NMDA-2A) ligands from natural products and provide subunit-selective drug candidates for Alzheimer's disease (AD). The recombinant NMDA-2A containing a tag of epidermal growth factor receptor (EGFR) was expressed in Escherichia coli cells and immobilized on ibrutinib-modified microspheres based on the specific reaction between EGFR and its inhibitor ibrutinib. A novel affinity stationary phase was synthesized to screen NMDA-2A ligands from Gardenia jasminoides Ellis. The immobilized receptor column exhibited excellent receptor selectivity and ligand-binding activity. Crocetin was screened by using this method. In a cellular model of AD, the protein level of NMDA-2A was significantly decreased compared with the control group, while treatment with crocetin significantly increased NMDA-2A level in a concentration-dependent manner, confirming that crocetin could bind to NMDA-2A in vitro. In the present study, we developed a reliable method for the rapid identification of NMDA-2A ligands from natural products, which may be used as a platform for new drug discovery to generate high-quality drug candidates.

MiR-29c-3p Attenuates β-Amyloid-Induced Neurotoxicity in Alzheimer’s Disease Through Regulating β-Site Amyloid Precursor Protein-Cleaving Enzyme 1

The aberrantly expressed microRNAs (miRNAs) including miR-29c-3p have been reported in the brains of Alzheimer's disease (AD) patients in recent researches. Nevertheless, the functional role and underlying molecular mechanism of miR-29c-3p in AD pathogenesis are still not well elucidated. The purpose of this study was to examine whether miR-29c-3p regulated β-Ameyloid (Aβ)-induced neurotoxicity by targeting β-site amyloid precursor protein-cleaving enzyme 1 (BACE1). The expressions of miR 29c 3p and BACE1 mRNA and protein levels in Aβ-treated PC12 cellular AD model were examined by qRT-PCR and western blot analyses. Luciferase reporter assay verified the potential target of miR 29c 3p. Cell viability, apoptosis, and caspase-3 activity in PC12 cells were detected by the MTT assay, flow cytometry, and caspase-3 activity assay, respectively. Our results indicated that miR-29c-3p downregulation and BACE1 upregulation existed in the cellular AD model of PC12 cells. Moreover, miR-29c-3p directly inhibited BACE1 expression. miR-29c-3p overexpression and BACE1 knockdown strengthened Aβ-induced cell apoptosis, and caspase-3 activity in PC12 cells, which was partially eliminated by over-expression of BACE1. Conversely, BACE1 knockdown reversed the miR-29c-3p inhibition- mediated inhibitory effect on Aβ-induced cell toxicity, apoptosis, and caspase-3 activity in PC12 cells. Considering, miR-29c-3p attenuated Aβ-induced neurotoxicity through targeting BACE1 in an cellular AD model of PC12, providing a potential therapeutic target for AD treatment.

Neuroprotective and Antioxidant Role of Oxotremorine-M, a Non-selective Muscarinic Acetylcholine Receptors Agonist, in a Cellular Model of Alzheimer Disease

Alzheimer disease (AD) is a multifactorial and age-dependent neurodegenerative disorder, whose pathogenesis, classically associated with the formation of senile plaques and neurofibrillary tangles, is also dependent on oxidative stress and neuroinflammation chronicization. Currently, the standard symptomatic therapy, based on acetylcholinesterase inhibitors, showed a limited therapeutic potential, whereas disease-modifying treatment strategies are still under extensive research. Previous studies have demonstrated that Oxotremorine-M (Oxo), a non-selective muscarinic acetylcholine receptors agonist, exerts neurotrophic functions in primary neurons, and modulates oxidative stress and neuroinflammation phenomena in rat brain. In the light of these findings, in this study, we aimed to investigate the neuroprotective effects of Oxo treatment in an in vitro model of AD, represented by differentiated SH-SY5Y neuroblastoma cells exposed to Aβ1-42 peptide. The results demonstrated that Oxo treatment enhances cell survival, increases neurite length, and counteracts DNA fragmentation induced by Aβ1-42 peptide. The same treatment was also able to block oxidative stress and mitochondria morphological/functional impairment associated with Aβ1-42 cell exposure. Overall, these results suggest that Oxo, by modulating cholinergic neurotransmission, survival, oxidative stress response, and mitochondria functionality, may represent a novel multi-target drug able to achieve a therapeutic synergy in AD.Graphical Illustration of the main pathological hallmarks and mechanisms underlying AD pathogenesis, including neurodegeneration and oxidative stress, efficiently counteracted by treatment with Oxo, which may represent a promising therapeutic molecule. Created with BioRender.com under academic license.

Rational Design of a Theranostic Agent Triggered by Endogenous Nitric Oxide in a Cellular Model of Alzheimer's Disease.

Oxidative damage caused by upregulated nitric oxide (NO) plays an important role in the pathogenesis of Alzheimer's disease (AD). Currently, stimulus-triggered theranostic agents have received much attention due to benefits on disease imaging and targeted therapeutic effects. However, the development of a theranostic agent triggered by NO for AD remains unexplored. Herein, through the mechanism analysis of the reaction between a fluorophore of 9,14-diphenyl-9,14-dihydrodibenzo[a,c]phenazine (DPAC) and NO, which we occasionally found and thereafter structure optimization of DPAC, a theranostic agent DPAC-(peg)4-memantine was fabricated. In an AD cellular model, DPAC-(peg)4-memantine exhibits NO sensing ability for AD imaging. Meanwhile, DPAC-(peg)4-memantine shows improved therapeutic by targeted drug release triggered by NO and sustained therapeutic effects owing to the synergetic antioxidative abilities via the anti-AD drug and NO scavenging. This work provides an unprecedented avenue for the studies on not only AD but also other diseases with NO upregulation.

Melatonin Attenuates Methamphetamine-Induced Alteration of Amyloid β Precursor Protein Cleaving Enzyme Expressions via Melatonin Receptor in Human Neuroblastoma Cells.

Alzheimer's disease (AD) is the most prominent neurodegenerative disease represented by the loss of memory and cognitive impairment symptoms and is one of the major health imperilments among the elderly. Amyloid (Aβ) deposit inside the neuron is one of the characteristic pathological hallmarks of this disease, leading to neuronal cell death. In the amyloidogenic processing, the amyloid precursor protein (APP) is cleaved by beta-secretase and γ-secretase to generate Aβ. Methamphetamine (METH) is a psychostimulant drug that causes neurodegeneration and detrimental cognitive deficits. The analogy between the neurotoxic and neurodegenerative profile of METH and AD pathology necessitates an exploration of the underlying molecular mechanisms. In the present study, we found that METH ineluctably affects APP processing, which might contribute to the marked production of Aβ in human neuroblastoma cells. Melatonin, an indolamine produced and released by the pineal gland as well as other extrapineal, has been protective against METH-induced neurodegenerative processes, thus rescuing neuronal cell death. However, the precise action of melatonin on METH has yet to be determined. We further propose to investigate the protective properties of melatonin on METH-induced APP-cleaving secretases. Pretreatment with melatonin significantly reversed METH-induced APP-cleaving secretases and Aβ production. In addition, pretreatment with luzindole, a melatonin receptor antagonist, significantly prevented the protective effect of melatonin, suggesting that the attenuation of the toxic effect on METH-induced APP processing by melatonin was mediated via melatonin receptor. The present results suggested that melatonin has a beneficial role in preventing Aβ generation in a cellular model of METH-induced AD.

Guanabenz mitigates the neuropathological alterations and cell death in Alzheimer's disease.

Alzheimer's disease (AD) pathology is characterized by cognitive impairment, increased acetylcholinesterase (AChE) activity, and impaired neuronal communication. Clinically, AChE inhibitors are being used to treat AD patients; however, these remain unable to prevent the disease progression. Therefore, further development of new therapeutic molecules is required having broad spectrum effects on AD-related various neurodegenerative events. Since repurposing is a quick mode to search the therapeutic molecules; henceforth, this study was conducted to evaluate the anti-Alzheimer activity of drug guanabenz which is already in use for the management of high blood pressure in clinics. The study was performed employing both cellular and rat models of AD along with donepezil as reference drug. Guanabenz treatment in both the experimental models showed significant protection against AD-specific behavioral and pathological indicators like AChE activity, tau phosphorylation, amyloid precursor protein, and memory retention. In conjunction, guanabenz also attenuated the AD-related oxidative stress, impaired mitochondrial functionality (MMP, cytochrome-c translocation, ATP level, and mitochondrial complex I activity), endoplasmic reticulum stress (GRP78, GADD153, cleaved caspase-12), neuronal apoptosis (Bcl-2, Bax, cleaved caspase-3), and DNA fragmentation. In conclusion, findings suggested the panoptic protective effect of guanabenz on disease-related multiple degenerative markers and signaling. Furthermore, clinical trial may shed light and expedite the availability of new therapeutic anti-Alzheimer's molecule for the wellbeing of AD patients.

Inhibition of Glycogen Synthase Kinase and the Neuroprotective Function of Conjugated ZnO-Osthol Nanoparticles in Alzheimer's Disease.

A critical factor in the cause and progression of Alzheimer's disease (AD) is the growth of β-amyloid peptide (Aβ) in the brain. The mechanism of this effect is still unknown, although the effect of osthol on Aβ-induced inflammation is neuroprotective in AD and supplementation with zinc might prevent or delay the onset of dementia. In the current study, by inducing APP vector in human BE (2)-M17 cells, we established a cellular model of AD and investigated the protective effect of osthol (7-methoxy-8-3-methyl-2-butenyl-2H-1-benzopyran-2-one)-zinc oxide nanoparticles. The osthol-conjugated zinc oxide nanoparticles could significantly increase cell viability by inhibiting cell apoptosis. Osthol treatment has also prevented synaptic proteins such as postsynaptic density-95 (PSD-95), synaptophysin (SYP), and synapsin-1 from decreasing in APP-induced BE (2)-M17 cells. In addition, the expression of miR-132 was significantly upregulated by osthol by triggering the Wnt/β-catenin signaling pathway. We conclude from our observations that osthol is a potential drug for the treatment of a neurodegenerative disease, Alzheimer's. The key reason was that by upregulating miR-132, osthol could inhibit APP expression to prevent AD from occurring.

From Kinase Inhibitors to Multitarget Ligands as Powerful Drug Leads for Alzheimer's Disease using Protein-Templated Synthesis.

Multitarget directed ligands (MTDLs) are arising as promising tools to tackle complex diseases. The main goal of this work is to create powerful modulating agents for neurodegenerative disorders. To achieve this aim, we have combined fragments that inhibit key protein kinases involved in the main pathomolecular pathways of Alzheimer's disease (AD) such as tau aggregation, neuroinflammation and decreased neurogenesis, whilst looking for a third action in beta‐secretase (BACE1), responsible of β‐amyloid production. We obtained well‐balanced MTDLs with in vitro activity in three different relevant targets and efficacy in two cellular models of AD. Furthermore, computational studies confirmed how these compounds accommodate adequately into the long and rather narrow BACE1 catalytic site. Finally, we employed in situ click chemistry using BACE1 as protein template as a versatile synthetic tool that allowed us to obtain further MTDLs.

From Kinase Inhibitors to Multitarget Ligands as Powerful Drug Leads for Alzheimer's Disease using Protein‐Templated Synthesis

AbstractMultitarget directed ligands (MTDLs) are arising as promising tools to tackle complex diseases. The main goal of this work is to create powerful modulating agents for neurodegenerative disorders. To achieve this aim, we have combined fragments that inhibit key protein kinases involved in the main pathomolecular pathways of Alzheimer's disease (AD) such as tau aggregation, neuroinflammation and decreased neurogenesis, whilst looking for a third action in beta‐secretase (BACE1), responsible of β‐amyloid production. We obtained well‐balanced MTDLs with in vitro activity in three different relevant targets and efficacy in two cellular models of AD. Furthermore, computational studies confirmed how these compounds accommodate adequately into the long and rather narrow BACE1 catalytic site. Finally, we employed in situ click chemistry using BACE1 as protein template as a versatile synthetic tool that allowed us to obtain further MTDLs.

Epigenetic transcripts of LINC01311 and hsa-miR-146a-5p regulate neural development in a cellular model of Alzheimer's disease.

Emerging evidence has shown that Long noncoding RNAs (LncRNAs) are aberrantly expressed and functionally involved in the development of neurodegenerative disorders. In this work, we investigated the regulatory effects of lncRNA of LINC01311 and its competing endogenous RNA target of hsa-miR-146a-5p in a cellular model of Alzheimer's disease (AD). SH-SY5Y cells were treated with synthetic Βeta-Amyloid Peptide (1-42) (AB1-42) in vitro to induce AD-like neural injuries. Expressions of LINC01311 and hsa-miR-146a-5p were monitored by qRT-PCR. LINC01311 was upregulated and hsa-miR-146a-5p downregulated to examine their functional regulations on AB1-42-induced apoptosis, proliferation slowdown, autophagy, and amyloid precursor protein (APP) accumulations. Hsa-miR-146a-5p was also overexpressed in LINC01311-upregulated SH-SY5Y cells to examine their correlated regulations on AB1-42-induced neural injuries. LINC01311 was downregulated whereas hsa-miR-146a-5p upregulated in AB1-42 treated SH-SY5Y cells. LINC01311 upregulation and hsa-miR-146a-5p downregulation protected AB1-42-induced apoptosis, proliferation slowdown, autophagy, and APP accumulations in SH-SY5Y cells. Hsa-miR-146a-5p overexpression reversed the protection of LINC01311 on AB1-42-induced neural injuries. Our work demonstrated that the epigenetic axis of LINC01311/hsa-miR-146a-5p was involved in the functional regulation of human-lineage neurons in a cellular model of AD, thus suggesting a clinical potential of exploring epigenetic network for treating AD patients.

MicroRNA-146a inhibition promotes total neurite outgrowth and suppresses cell apoptosis, inflammation, and STAT1/MYC pathway in PC12 and cortical neuron cellular Alzheimer's disease models.

This study aimed to explore the effect of microRNA (miR)-146a inhibition on regulating cell apoptosis, total neurite outgrowth, inflammation, and STAT1/MYC pathway in Alzheimer's disease (AD). PC12 and cortical neuron cellular AD models were constructed by Aβ1-42 insult. For the former model, nerve growth factor (NGF) stimulation was previously conducted. miR-146a inhibitor and negative-control (NC) inhibitor were transfected into the two cellular AD models, and then cells were named miR-inhibitor group and NC-inhibitor group, respectively. After transfection, cell apoptosis, total neurite outgrowth, supernatant inflammation cytokines, and STAT1/MYC pathway were detected. miR-146a expression was similar between PC12 cellular AD model and control cells (NGF-stimulated PC12 cells), while miR-146a expression was increased in cortical neuron cellular AD model compared with control cells (rat embryo primary cortical neurons). In both PC12 and cortical neuron cellular AD models, miR-146a expression was reduced in miR-inhibitor group compared with NC-inhibitor group after transfection. Furthermore, cell apoptosis was attenuated, while total neurite outgrowth was elevated in miR-inhibitor group compared with NC-inhibitor group. As for supernatant inflammatory cytokines, tumor necrosis factor-α, interleukin (IL)-1β, IL-6, and IL-17 levels were lower in miR-inhibitor group than in NC-inhibitor group. Additionally, STAT1 and c-Myc mRNA and protein expressions were attenuated in miR-inhibitor group compared with NC-inhibitor group. In conclusion, miR-146a potentially represented a viable therapeutic target for AD.

Targeting MicroRNA-125b Promotes Neurite Outgrowth but Represses Cell Apoptosis and Inflammation via Blocking PTGS2 and CDK5 in a FOXQ1-Dependent Way in Alzheimer Disease.

This study aimed to explore the molecular regulatory network among microRNA-125b (miR-125b), forkhead box Q1 (FOXQ1), prostaglandin-endoperoxide synthase 2 (PTGS2), and cyclin-dependent kinase 5 (CDK5), as well as their effects on cell apoptosis, neurite outgrowth, and inflammation in Alzheimer disease (AD). Rat embryo cerebral cortex neurons and nerve growth factor–stimulated PC12 cells were insulted by Aβ1−42 to construct two AD cellular models. Negative control (NC) inhibitor, miR-125b inhibitor, NC siRNA, FOXQ1 siRNA, PTGS2 siRNA, and CDK5 siRNA were transferred into the two AD cellular models alone or combined. Then, cell apoptosis, neurite outgrowth, proinflammatory cytokines, miR-125b, FOXQ1, PTGS2, and CDK5 expressions were detected. MiR-125b inhibition facilitated neurite outgrowth but suppressed cell apoptosis and proinflammatory cytokines (tumor necrosis factor-α, interleukin 1β, and interleukin 6); meanwhile, it upregulated FOXQ1 but downregulated PTGS2 and CDK5. Furthermore, FOXQ1 inhibition promoted cell apoptosis and proinflammatory cytokines but repressed neurite outgrowth; PTGS2 inhibition achieved the opposite effects; CDK5 inhibition attenuated cell apoptosis, whereas it less affected neurite outgrowth and inflammation. Notably, FOXQ1 inhibition attenuated, whereas PTGS2 inhibition elevated the effect of miR-125b inhibition on regulating neurite outgrowth, cell apoptosis, and proinflammatory cytokines. As for CDK5 inhibition, it enhanced the effect of miR-125b inhibition on regulating cell apoptosis, but less impacted the neurite outgrowth and proinflammatory cytokines. Additionally, PTGS2 inhibition and CDK5 inhibition both reversed the effect of FOXQ1 inhibition on regulating cell apoptosis, neurite outgrowth, and proinflammatory cytokines. In conclusion, targeting miR-125b alleviates AD progression via blocking PTGS2 and CDK5 in a FOXQ1-dependent way.

DNA Double-Strand Break Accumulation in Alzheimer's Disease: Evidence from Experimental Models and Postmortem Human Brains.

Alzheimer's disease (AD) is a progressive neurodegenerative disease that accounts for a majority of dementia cases. AD is characterized by progressive neuronal death associated with neuropathological lesions consisting of neurofibrillary tangles and senile plaques. While the pathogenesis of AD has been widely investigated, significant gaps in our knowledge remain about the cellular and molecular mechanisms promoting AD. Recent studies have highlighted the role of DNA damage, particularly DNA double-strand breaks (DSBs), in the progression of neuronal loss in a broad spectrum of neurodegenerative diseases. In the present study, we tested the hypothesis that accumulation of DNA DSB plays an important role in AD pathogenesis. To test our hypothesis, we examined DNA DSB expression and DNA repair function in the hippocampus of human AD and non-AD brains by immunohistochemistry, ELISA, and RT-qPCR. We observed increased DNA DSB accumulation and reduced DNA repair function in the hippocampus of AD brains compared to the non-AD control brains. Next, we found significantly increased levels of DNA DSB and altered levels of DNA repair proteins in the hippocampus of 5xFAD mice compared to non-transgenic mice. Interestingly, increased accumulation of DNA DSBs and altered DNA repair proteins were also observed in cellular models of AD. These findings provided compelling evidence that AD is associated with accumulation of DNA DSB and/or alteration in DSB repair proteins which may influence an important early part of the pathway toward neural damage and memory loss in AD.

The Alzheimer's disease‐associated C99 fragment of APP regulates cellular cholesterol trafficking

The link between cholesterol homeostasis and cleavage of the amyloid precursor protein (APP), and how this relationship relates to Alzheimer's disease (AD) pathogenesis, is still unknown. Cellular cholesterol levels are regulated through crosstalk between the plasma membrane (PM), where most cellular cholesterol resides, and the endoplasmic reticulum (ER), where the protein machinery that regulates cholesterol levels resides. The intracellular transport of cholesterol from the PM to the ER is believed to be activated by a lipid‐sensing peptide(s) in the ER that can cluster PM‐derived cholesterol into transient detergent‐resistant membrane domains (DRMs) within the ER, also called the ER regulatory pool of cholesterol. When formed, these cholesterol‐rich domains in the ER maintain cellular homeostasis by inducing cholesterol esterification as a mechanism of detoxification while attenuating its de novo synthesis. In this manuscript, we propose that the 99‐aa C‐terminal fragment of APP (C99), when delivered to the ER for cleavage by γ‐secretase, acts as a lipid‐sensing peptide that forms regulatory DRMs in the ER, called mitochondria‐associated ER membranes (MAM). Our data in cellular AD models indicates that increased levels of uncleaved C99 in the ER, an early phenotype of the disease, upregulates the formation of these transient DRMs by inducing the internalization of extracellular cholesterol and its trafficking from the PM to the ER. These results suggest a novel role for C99 as a mediator of cholesterol disturbances in AD, potentially explaining early hallmarks of the disease.

Liraglutide Attenuates Aβ42 Generation in APPswe/SH-SY5Y Cells Through the Regulation of Autophagy.

ObjectiveThis study aimed to clarify whether liraglutide, a GLP-1 analogue, can ameliorate Aβ pathology through the regulation of autophagy in Alzheimer’s disease (AD) and to explore the related mechanisms thereof.MethodsWe used SH-SY5Y cells transiently transfected with APP695swe plasmid as an AD cellular model. Transfected cells were treated with liraglutide for 24 h in the presence or absence of 3-MA. Autophagy markers and the Aβ level were then evaluated by Western blot and ELISA. We also investigated the potential involvement of mTOR and JNK pathway in liraglutide-mediated autophagy.ResultsOur results showed that liraglutide reduced Aβ42 generation and enhanced autophagy in APPswe/SH-SY5Y cells; however, these effects could be counteracted with 3-MA. Furthermore, our data showed that liraglutide-induced autophagy does not follow the mTOR pathway. Liraglutide might promote autophagy in APPswe/SH-SY5Y cells by activating the JNK pathway and inhibiting the beclin-1/bcl-2 complex.ConclusionHere, we report a novel mechanism underlying liraglutide-attenuated Aβ42 generation through the activation of autophagy in AD cellular model.

Feasibility and Efficacy of Intra-Arterial Administration of Embryonic Stem Cell Derived-Mesenchymal Stem Cells in Animal Model of Alzheimer's Disease.

Mesenchymal stem cells (MSCs) promote functional recoveries in pathological experimental models of the central nervous system and are currently being tested in clinical trials for neurological disorders. However, no studies have examined the various roles of embryonic stem cell derived (ES)-MSCs in eliciting therapeutic effects for Alzheimer's disease (AD). In the present study, we investigated the neuroprotective effect of ES-MSCs in cellular and animal models of AD, as well as the safety of the intra-arterial administration of ES-MSCs in an AD animal model. ES-MSCs displayed higher cell viability than that of bone marrow (BM)-MSCs in amyloid-β (Aβ)-induced cellular models. Moreover, the efficacy of autophagy induction in ES-MSCs was comparable to that of BM-MSCs; however, intracellular Aβ levels were more significantly reduced in ES-MSCs than in BM-MSCs. In a rat model of AD, ES-MSCs significantly inhibited Aβ-induced cell death in the hippocampus and promoted autophagolysosomal clearance of Aβ, which was concomitantly followed by decreased levels of Aβ in the hippocampus. Furthermore, ES-MSC treatment in Aβ-treated rats featured a higher memory performance than that of rats injected solely with Aβ. Finally, intra-arterial administration of an appropriate cell density of ES-MSCs was safe and free from in situ occlusion or cerebral ischemia. These data support the therapeutic potential of ES-MSCs and clinical applications of the intra-arterial route of ES-MSC administration in AD.

Long non-coding RNA ANRIL knockdown suppresses apoptosis and pro-inflammatory cytokines while enhancing neurite outgrowth via binding microRNA-125a in a cellular model of Alzheimer's disease.

The present study aimed to investigate the effect of the long non-coding RNA antisense non-coding RNA in the INK4 locus (lnc-ANRIL) knockdown on apoptosis, neurite outgrowth and inflammation based on a PC12 cellular Alzheimer's disease (AD) model. A cellular AD model was constructed by treating nerve growth factor stimulated PC12 cells with amyloid β (Aβ) 1–42 and then control knockdown plasmid and lnc-ANRIL knockdown plasmid were transfected in the PC12 cellular AD model as the KD- negative control (NC) group or the AD-ANRIL group respectively. Apoptosis, neurite outgrowth, pro-inflammatory cytokines and microRNA (miR)-125a were assessed. Rescue experiments were conducted by transfecting lnc-ANRIL knockdown plasmid and lnc-ANRIL knockdown plasmid and miR-125a inhibitor in the PC12 cellular AD model as the KD-ANRIL group or KD-ANRIL + KD-miR-125a group respectively. Following transfection, cell apoptosis deccreased while neurite outgrowth increased in the KD-ANRIL group compared with the KD-NC group (all P<0.01). Concerning inflammation, tumor necrosis factor-α (TNF-α) and interleukin (IL)-1β, IL-6 and IL-17 were decreased in the KD-ANRIL group compared with the KD-NC group (all P<0.01). miR-125a was negatively regulated by lnc-ANRIL and therefore rescue experiments were subsequently conducted. In the rescue experiments, cell apoptosis was increased while total neurite outgrowth was inhibited in the KD-ANRIL + KD-miR-125a group compared with the KD-ANRIL group (all P<0.01), and TNF-α, IL-1β, IL-6 and IL-17 were increased in the KD-ANRIL + KD-miR-125a group compared with the KD-ANRIL group (all P<0.01). A luciferase reporter assay demonstrated that lnc-ANRIL directly bound miR-125a. lnc-ANRIL knockdown suppressed cell apoptosis and inflammation while promoting neurite outgrowth via binding of miR-125a in AD.

Neuroprotective effects of Fomes officinalis Ames polysaccharides on Aβ25-35-induced cytotoxicity in PC12 cells through suppression of mitochondria-mediated apoptotic pathway.

Aggregation of Aβ is a pathological hallmark of Alzheimer's disease (AD). The purpose of this study was to identify the protective roles of different polysaccharide components in Fomes officinalis Ames polysaccharides (FOAPs) against Aβ25-35-induced neurotoxicity in PC12 cells. Different doses of FOAPs components (i.e. FOAPs-a and FOAPs-b) were added to PC12 cells about 2h before β-amyloid protein fragment 25-35 (Aβ25-35) exposure. The AD cellular model of PC12 cells was established using Aβ25-35. Then the PC12 cells were divided into 9 groups including: control group, Donepezil hydrochloride (DHCL) group, model group treated using 40μM Aβ25-35, followed by FOAPs-a and FOAPs-b interference (50, 100 and 200μg/mL). The mitochondrial reactive oxygen species (ROS), ATP, superoxide dismutase (SOD), malondialdehyde (MDA), lactate dehydrogenase (LDH) and mitochondrial membrane potential (MMP) were determined by commercial kits. The Cytochrome C, Bcl-2 and Bax expressions in the mitochondria and cytosol was determined by using Western blot analysis. FOAPs-a and FOAPs-b could significantly inhibit the LDH release, MDA level and the over accumulation of ROS induced by Aβ25-35 in PC12 cells in a dose-dependent manner. They could also effectively prevent Aβ25-35-stimulated cytotoxicity, which involved in attenuating cell apoptosis, increasing the ratio of Bcl-2/Bax and inhibiting Cytochrome C release from mitochondria to cytosol in PC12 cells. Moreover, FOAPs-a and FOAPs-b significantly alleviated mitochondrial dysfunction by regulating the MMP, as well as promoting the mitochondrial ATP synthesis. FOAPs-a and FOAPs-b played neuroprotective roles against Aβ25-35-induced cytotoxicity in PC12 cells through suppressing the mitochondria-mediated apoptotic pathway.