Alzheimer's Disease Cell Model Research Articles

Curcumin Alleviates Aβ42-Induced Neuronal Metabolic Dysfunction via the Thrb/SIRT3 Axis and Improves Cognition in APPTG Mice.

Curcumin has been reported to have a therapeutic effect on Alzheimer's disease (AD), but the specific mechanism remains to be elucidated. In the present research, we aimed to investigate the effect and molecular mechanism of curcumin on AD. Mouse primary hippocampal neuron cells were treated with various concentrations of beta-amyloid 42 (Aβ42) and the results found that Aβ42 inhibited cell viability in a dose-dependent manner. Compared with 50ng/mL Aβ42, 500ng/mL Aβ42 could further promote cell apoptosis, reduce the ratio of Nicotinamide adenine dinucleotide (NAD(+))/Nicotinamide adenine diphosphate hydride (NADH) and Adenosine 5'-triphosphate (ATP) level, and inhibit Sirtuins 3 (SIRT3) deacetylation activity and protein expression of Thyroid hormone receptor beta (Thrb) and SIRT3. Hence, 500ng/mL Aβ42 was used to establish a cell model of AD. Curcumin significantly reversed the inhibitory effects of Aβ42 on cell viability, SIRT3 deacetylation activity, the ratio of NAD+/NADH, ATP level and the protein expression of Thrb and SIRT3, and the promotive effect on apoptosis. ChIPBase was used to predict the binding region of Thrb and SIRT3. Dual luciferase reporter gene and Chromatin immune precipitation (ChIP) assays were employed to verify the relationship between Thrb and promoter of SIRT3 mRNA. Overexpression of Thrb recovered Aβ42 induced metabolic dysfunction, while Thrb silence aggravated Aβ42 induced metabolic dysfunction. Moreover, Thrb silence or 3-TYP (a selective inhibitor of SIRT3) treatment abolished the amelioration of curcumin on Aβ42 induced metabolic dysfunction. Additionally, curcumin attenuated memory deficits in Amyloid precursor protein transgenic (APPTG) mice. Collectively, curcumin alleviated Aβ42-induced neuronal metabolic dysfunction through increasing Thrb expression and SIRT3 activity and improved cognition in APPTG mice.

Functional effects of berberine in modulating mitochondrial dysfunction and inflammatory response in the respective amyloidogenic cells and activated microglial cells – In vitro models simulating Alzheimer's disease pathology

AimBerberine (BBR) is an alkaloid extracted from Coptidis Rhizoma, also known as Huang-Lian. Huang-Lian has been used extensively in traditional Chinese medicine for the treatment of various diseases, including diabetes and dementia. Because Alzheimer's disease (AD) is a complex disease that involves various pathophysiological changes, the diverse neuroprotective effects of BBR may be useful for improving the brain's energy state at an early stage of the disease. Main methodsWe performed extracellular flux and 1H NMR-based metabolic profiling analyses to investigate the effects of BBR on metabolic processes in these cells. Pioglitazone (PIO), a peroxisome proliferator-activated receptor-γ (PPARγ) agonist has been studied extensively for the treatment of AD. We explored the combination dosing effects of BBR and PIO in vitro, then leveraged computational methods to explain the experimental finding. Key findingsBBR demonstrates potential in modulating the mitochondrial bioenergetics and attenuating dysfunction of the primary energy and glutathione metabolism pathways in an AD cell model. It also suppresses basal respiration and reduces the production of pro-inflammatory cytokines in activated microglial cells. Both experimental and computational observations indicate that BBR and PIO have comparable binding affinities to the PPARγ protein, suggesting both drugs may have some overlapping effects for AD. SignificanceBBR exerts beneficial effects on disrupted metabolic processes in amyloidogenic cells and activated microglial cells, which are important for preventing or delaying early-stage disease progression. The choice of BBR or PIO for AD treatment depends on their respective pharmacokinetic profiles, delivery, efficacy and safety, and warrants further study.

Formulated Chinese medicine Shaoyao Gancao Tang reduces NLRP1 and NLRP3 in Alzheimer's disease cell and mouse models for neuroprotection and cognitive improvement.

Amyloid β (Aβ) plays a major role in the neurodegeneration of Alzheimer’s disease (AD). The accumulation of misfolded Aβ causes oxidative stress and inflammatory damage leading to apoptotic cell death. Traditional Chinese herbal medicine (CHM) has been widely used in treating neurodegenerative diseases by reducing oxidative stress and neuroinflammation. We examined the neuroprotective effect of formulated CHM Shaoyao Gancao Tang (SG-Tang, made of Paeonia lactiflora and Glycyrrhiza uralensis at 1:1 ratio) in AD cell and mouse models. In Aβ-GFP SH-SY5Y cells, SG-Tang reduced Aβ aggregation and reactive oxygen species (ROS) production, as well as improved neurite outgrowth. When the Aβ-GFP-expressing cells were stimulated with conditioned medium from interferon (IFN)-γ-activated HMC3 microglia, SG-Tang suppressed expressions of inducible nitric oxide synthase (iNOS), NLR family pyrin domain containing 1 (NLRP1) and 3 (NLRP3), tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6, attenuated caspase-1 activity and ROS production, and promoted neurite outgrowth. In streptozocin-induced hyperglycemic APP/PS1/Tau triple transgenic (3×Tg-AD) mice, SG-Tang also reduced expressions of NLRP1, NLRP3, Aβ and Tau in hippocampus and cortex, as well as improved working and spatial memories in Y maze and Morris water maze. Collectively, our results demonstrate the potential of SG-Tang in treating AD by moderating neuroinflammation.

Progesterone Suppresses Cholesterol Esterification in APP/PS1 mice and a cell model of Alzheimer’s Disease

AimsCholesteryl ester(CE), generated from the mitochondria associated membrane (MAM), is involved in the pathogenesis of Alzheimer's Disease (AD). In theory, the different neuroprotective effects of progesterone in AD are all linked to MAM, yet the effect on cholesterol esterification has not been reported. Therefore, this study was aimed to investigate the regulation of progesterone on intracerebral CE in AD models and the underlying mechanism. MethodsAPP/PS1 mice and AD cell model induced by Aβ 25-35 were selected as the research objects. APP/PS1 mice were daily administrated intragastrically with progesterone and The Morris Water Maze test was performed to detect the learning and memory abilities. Intracellular cholesterol was measured by Cholesterol/Cholesteryl Ester Quantitation Assay. The structure of MAMs were observed with transmission electron microscopy. The expression of acyl-CoA: cholesterol acyltransferase 1 (ACAT1), ERK1/2 and p-ERK1/2 were detected with western blotting, immunohistochemistry or immunofluorescence. ResultsProgesterone suppressed the accumulation of intracellular CE, shortened the length of abnormally prolonged MAM in cortex of APP/PS1 mice. Progesterone decreased the expression of ACAT1, which could be blocked by progesterone receptor membrane component 1 (PGRMC1) inhibitor AG205. The ERK1/2 pathway maybe involved in the progesterone mediated regulation of ACAT1 in AD models, rather than the PI3K/Akt and the P38 MEPK pathways. SignificanceThe results supported a line of evidence that progesterone regulates CE level and the structure of MAM in neurons of AD models, providing a promising treatment against AD on the dysfunction of cholesterol metabolism.

Regulation of Aβ40 and Aβ42 by formaldehyde exposure in a three‐dimensional (3D) human neural cell culture model for Alzheimer's Disease

Alzheimer's disease (AD), a chronic neurodegenerative disorder characterized by rapidly progressive cognitive dysfunction and behavior impairment, is the most common form of dementia which accounts for 60 to 70 percent of cases. Pathologically, AD is characterized by severe atrophy and neuronal loss, extracellular neuritic plaques composed mainly of aggregated amyloid-b (Ab), and intracellular neurofibrillary tangles consisting of hyperphosphorylated tau protein. Multilevel complex interactions between genetic, epigenetic, and environmental factors contribute to the occurrence and progression of AD. Formaldehyde (FA) is the simplest saturated aldehyde which is one of the most typical environmental and occupational aldehyde pollutants. Recent studies suggested that elevation of FA in the brain, blood or urine by endogenous and/or exogenous exposure may play important roles in AD development. A three-dimensional (3D) human neural cell culture model for AD which can recapitulate key events of AD pathology including β-amyloid plaques and neurofibrillary tangles was used to investigate the effect of FA exposure in AD development and progression in human cells. Immortalized human neural progenitor cells (ReNcell VM (ReN)) were virally transfected with AD-related mutant genes including mutant APP and/or PSEN1 and enriched based on GFP and/or mCherry signals by Fluorescence-activated cell sorting (FACS). In order to mimic the restrictive environment of human brain and allow Aβ deposition and aggregation, the FACS-sorted ReN cells were differentiated into neurons and maintained in a 3D Matrigel culture system. The AD Model and primary ReN cells were treated with different concentrations of FA as well as vehicle controls for 16 weeks. Expression of Aβ40 and Aβ42 were measured in 4, 6, 12 16 weeks by ELISA and cytotoxicity was evaluated by LDH activity assay. The results revealed that FA significantly elevates expression of Aβ40 and Aβ42 in a concentration-dependent manner both in AD model and ReN cells. It indicated that FA exposure may play a critical role in AD development and progression.

Vitamin D Attenuates Alzheimer-like Pathology Induced by Okadaic Acid.

Many elderly individuals suffer from Alzheimer's disease (AD), which causes a growing concern. We investigated the mechanism underlying the effects of vitamin D (VD) as a prophylactic treatment. A mouse model of okadaic-acid-induced AD-like pathology was used in vivo and in vitro. Morris water maze and field trials were used to assess cognitive function. The expression levels of VDR, MTHFR, LCMT-1, PP2A, p-TAU (Thr396), and T-TAU and the methylation level of PP2A were measured by Western blotting, and a reversal of the increase in the levels of these proteins in an AD cell model was observed. We used MTHFR-knockdown SH-SY5Y cells to further test the effects of VD, treated these cells with cycloheximide and MG132, and used RT-PCR to explore the mechanism underlying MTHFR targeting. We found that the effects of VD on AD were impaired by MTHFR knockdown through a pretranscriptional mechanism. In addition, VD attenuated AD-induced cognitive impairment and significantly suppressed the expression of TAU. Our findings indicated that VD treatment alleviated TAU accumulation and rescued methylated PP2A by increasing the expression of LCMT-1 and MTHFR.

Amyloid precursor protein intra-cellular domain (AICD), Aβ and their confounding synergistic effects differentially regulate the degradome of cellular models of Alzheimer's disease

Altered expressions of protein-coding genes and microRNAs have been implicated in the pathogenesis of Alzheimer's disease (AD). The disrupted miRNA landscape (degradome) has been studied in AD mainly from the Aβ perspective. That Amyloid Precursor Protein C- terminal Domain (AICD) is involved in altering the cellular transcriptome has been reported, but its role in the degradome is still unexplored. The involvement of other long non-coding RNAs (lncRNA) is being realized recently. Using small RNA sequencing from an AD cell model, we observed perturbations in the levels of 47 miRNAs deregulated between the control and Aβ groups, and 26 deregulated miRNAs between the control and AICD + Aβ (AD) groups. Additionally, using a novel bioinformatics pipeline, we obtained a total of 263 differentially expressed lncRNAs in Aβ versus control group, and 41 deregulated lncRNAs in the AD versus control group. Effect of Aβ and AICD, individually and in combination, were validated with top regulated miRNA hits. Several of these miRNAs were found to target key Receptor Tyrosine Kinase (RTK) - many of which are implicated in AD. Understanding the total cellular non-coding transcriptome in the context of AD is likely to open up new putative targets for the disease intervention.

MicroRNA-340-5p increases telomere length by targeting telomere protein POT1 to improve Alzheimer's disease in mice.

Alzheimer's disease (AD) is a chronic neurodegenerative disorder which is the primary cause of dementia in the elderly. Telomere attrition has been proposed as a hallmark of aging. Our study aimed to explore the mechanism of the protection of telomere 1 (POT1) in regulating telomere length and affecting cellular senescence in AD. The AD mouse model was established by d-galactose and aluminum chloride,and the water maze test and dark avoidance test were used to detect the behaviors of mice and confirm the success of AD mouse model. AD cell model was established with HT22 cells induced by Aβ42 oligomers. POT1 expression in the AD model was detected by quantitative real-time polymerase chain reaction. Cellular telomere length in hippocampal tissue was analyzed by telomere restriction fragment. Localization of intracellular POT1, telomerase, and telomeres was analyzed by immunofluorescence and fluorescence in situ hybridization. Dual-luciferase assay was used to validate the targeted binding relationship between microRNA-340-5p(miR-340-5p) and POT1. After inhibiting POT1 expression, the symptoms of AD in mice were improved. Aβ1-42 deposition was reduced, whereas telomere length and telomerase activity was increased. Dual-luciferase assay verified the binding relationship between miR-340-5p and POT1. An increase in miR-340-5p expression could alleviate cellular senescence and AD symptoms. miR-340-5p increased cellular telomere length and delayed cell senescence by inhibiting POT1 expression to improve AD symptoms. This study made a conclusion that miR-340-5p increased cellular telomere length and delayed cell senescence by inhibiting POT1 expression to improve AD symptoms in mice.

TTP488 ameliorates NLRP3-associated inflammation, viability, apoptosis, and ROS production in an Alzheimer's disease cell model by mediating the JAK1/STAT3/NFκB/IRF3 pathway.

Alzheimer's disease (AD), the most prevalent dementia, is identified as a neurodegenerative disease arising from a degenerative disturbance in the central nervous system. A previous study reported that TTP488 could ameliorate symptoms in patients with mild AD, but the underlying mechanisms need to be studied further. Therefore, the objective of this study was to explore the role of TTP488 in the development of an AD cell model. Administration of TTP448 in an AD cell model reduced the expression of pro-inflammatory cytokines [interleukin (IL)-1β, IL-6, and TNF-α], reversed the inhibitory role of Aβ on cell proliferation and viability, and decreased Aβ-triggered cell apoptosis and reactive oxygen species (ROS) production. Furthermore, Aβ treatment induced activation of JAK1/STAT3/NFκB/IRF3 pathway as well as NLRP3 expression, and TTP488 administration partially reversed the activation of this pathway and NLRP3 expression. Use of WP1160, a STAT3 agonist, re-activated the downstream STAT3/NFκB/IRF3 pathway and NLRP3 expression. Moreover, we found that WP1160 counteracted the role of TTP488 in Aβ-induced SH-SY5Y cells' viability, inflammation, apoptosis, and ROS production. SIGNIFICANCE OF THE STUDY: This study explores the role of TTP488 in the development of an Alzheimer's disease (AD) cell model and confirms that TTP488 administration notably promotes cell proliferation and reduces apoptosis, inflammatory factor expression, and reactive oxygen species generation. Further, this study suggests that the NLRP3-relevant JAK1/STAT3/P65/IRF3 signalling pathway is related to AD pathogenesis.

Role of miR-211 in a PC12 cell model of Alzheimer's disease via regulation of neurogenin 2.

What is the central question of this study? What is the mechanism of miR-211 in an Alzheimer's disease cell model? What is the main finding and its importance? miR-211 was upregulated in an Alzheimer's disease cell model. It targeted neurogenin 2, reduced the activation of the phosphoinositide 3-kinase-Akt signalling pathway, inhibited the proliferation of the Alzheimer's disease cell model and promoted apoptosis. MicroRNAs (miRs) are aberrantly expressed in Alzheimer's disease (AD) patients. This study was intended to investigate the effect of miR-211 on an AD cell model and the involvement of neurogenin 2 (Ngn2). The appropriate dose and time for the effect of Aβ1-42 on PC12 cells were determined to establish an AD cell model. An effect of miR-211 expression on cell viability, proliferation and apoptosis was detected after cell transfection. Online prediction and a dual luciferase reporter gene assay were utilized to confirm the binding sequence of miR-211 and Ngn2. qRT-PCR and western blot analysis were applied to measure Ngn2 expression. A gain and loss of function assay of miR-211 and Ngn2 was performed, and activation of the phosphoinositide 3-kinase (PI3K)-Akt signaling pathway was detected. The AD cell model was induced by Aβ1-42 treatment. miR-211 expression was significantly enhanced after miR-211 transfection, leading to suppressed proliferation and promotion of apoptosis in Aβ1-42 -treated PC12 cells. In addition, miR-211 could downregulate Ngn2 mRNA and protein expression, while overexpression of Ngn2 could reverse the effects of miR-211 on Aβ1-42 -treated PC12 cells and significantly enhance the phosphorylated Akt and PI3K protein levels. miR-211 could inhibit growth of PC12 cells by suppressing Ngn2 expression and inactivating the PI3K-Akt signalling pathway.

Quercetin-3-O-Glucuronide Alleviates Cognitive Deficit and Toxicity in Aβ1-42 -Induced AD-Like Mice and SH-SY5Y Cells.

Alzheimer's disease (AD) is characterized by amyloid-β (Aβ) related imbalance, Tau-hyperphosphorylation, and neuroinflammation, in which Aβ and neuroinflammation can induce brain insulin resistance (IR). Gut microbiome disorder is correlated with inflammation in AD. As of yet, there are no effective treatments clinically. Thus, it is focused on the potential benefit of quercetin-3-O-glucuronide (Q3G), a pharmacologically active flavonol glucuronide, on AD treatment by regulating brain IR and the gut microbiome. AD mice model built through intracerebroventricular injection of Aβ1-42 and AD cell model developed through the SH-SY5Y cell line and Aβ1-42 are used to explore the protective effects of Q3G on AD. Neurobehavioral test, brain insulin signaling pathway, and high-throughput pyrosequencing of 16S rRNA are assessed. Data show that Q3G attenuates neuroinflammation and brain IR in Aβ1-42 -injected mice and relieves apoptosis in Aβ1-42 -treated SH-SY5Y cells by interrupting the downstream insulin signaling. Q3G ameliorates Aβ accumulation and Tau phosphorylation, restores CREB and BDNF levels in the hippocampus , and reverses Aβ1-42 -induced cognitive impairment. Besides, Q3G restores Aβ1-42 -induced reduction of short-chain fatty acids (SCFAs) and gut microbiota dysbiosis. Q3G can alleviate brain IR through directly acting on the brain or modulating the gut-brain axis, ultimately to relieve Aβ1-42 -induced cognitive dysfunction.

Icaritin and icariin reduce p-Tau levels in a cell model of Alzheimer's disease by downregulating glycogen synthase kinase 3β.

This study aimed to explore the neuroprotective effect of icariin/icaritin (ICA/ICT) and the role of ICA/ICT in the treatment of Alzheimer's disease (AD). ICA and ICT were used to treat okadaic acid (OA)-induced Tau hyperphosphorylation in SH-SY5Y cells. We detected the relative changes in Tau, p-Tau, protein phosphatase 2A (PP2A), and glycogen synthase kinase 3β (GSK-3β) by Western blotting and enzyme-linked immunosorbent assay. At 40nmol/L OA, the cell viability of the SH-SY5Y cells was significantly changed. We used different concentrations of ICA and IC to treat AD model cells and found that the effect of 2.5μmol/L ICA and 1 μmol/L ICT was best after 48H of treatment. After SH-SY5Y cell induction, the p-Tau levels were increased (P<0.05); after the ICA/ICT treatment, the p-Tau and GSK-3β levels were decreased (P<0.05), although PP2A expression did not change (P>0.05). We found that ICA and ICT exert an effect on AD model cells by decreasing the levels of GSK-3β and p-Tau. The therapeutic effect of ICT is slightly better than that of ICA. Although these drugs were effective in the cell model, more studies are required to determine whether they are promising for the treatment and prevention of AD.

Network pharmacology-based study of the therapeutic mechanism of resveratrol for Alzheimer's disease

To investigate the therapeutic mechanism of resveratrol (RES) for Alzheimer's disease (AD) in light of network pharmacology. We searched PubChem, BATMAN-TCM, Genecards, AD, TTD, String 11.0, AlzData, SwissTargetPrediction, Metascape and other databases for the therapeutic targets of RES and human AD-related targets. The intersection was determined using Venny 2.1 to obtain the therapeutic targets of RES for AD. The protein-protein interaction (PPI) network was constructed, the gene ontology (GO) was enriched and the Kyoto Encyclopedia of Genes and Genomes pathway (KEGG pathway) were analyzed. Cytoscape 3.7.1 software was used to construct a target-signaling pathway network of RES in the treatment of AD. Molecular docking verification was carried out on SwissDock (http://www.swissdock.ch/docking). We examined a 293Tau cell model of AD for changes in protein levels of pS396, pS199, Tau5, CDK5, glycogen synthase kinase 3β (GSK3β) and p-GSK3β in response to RES treatment using Western blotting. We obtained 182 targets of RES, 525 targets related to AD, and 36 targets of RES for AD treatment, among which 34.6% of the targets were protein-modifying enzymes, 27.7% were metabolite invertase, 13.8% were gene-specific transcriptional regulators, and 10.3% were transporters. The core key targets of RES in the treatment of AD included INS, APP, ESR1, MMP9, IGF1R, CACNA1C, MAPT (microtubule- associated protein Tau), MMP2, TGFB1 and GSK3B. Enrichment analysis of GO biological process suggested that the biological function of RES in AD treatment mainly involved the response to β-amyloid protein, positive regulation of transferase activity, the transmembrane receptor protein tyrosine kinase signaling pathway, regulation of behavior, learning or memory, aging, and transmembrane transport. KEGG pathway enrichment analysis showed that the most significantly enriched signaling pathways were AD pathway, PI3K-AKT signaling pathway, cGMP-PKG signaling pathway, and MAPK signaling pathway. Molecular docking results showed that RES had strong binding with ESR1, GSK3B, MMP9, IGF1R, APP and INS. In the cell model of AD, treatment with 50 μmol/L RES for 12 h significantly reduced the levels of pS396 and pS199 by regulating CDK5 and GSK3β activity (P < 0.001). RES produces therapeutic effects on AD by acting on multiple targets and affecting multiple signaling pathways and improves AD-associated pathologies via a direct action on Aβ and Tau pathological processes.

Caspase-1/IL-1\u03b2 represses membrane transport of GluA1 by inhibiting the interaction between Stargazin and GluA1 in Alzheimer\u2019s disease

BackgroundAlzheimer's disease is a neurodegenerative disease. Previous study has reported that caspase-1/IL-1β is closely associated with Alzheimer's disease. However, the biological role of caspase-1/IL-1β in Alzheimer's disease has not been fully elucidated. This study aimed to explore the mechanism of action of caspase-1/IL-1β in Alzheimer's disease.MethodsMouse hippocampal neurones were treated with Aβ1-42 to induce Alzheimer's disease cell model. APP/PS1 mice and Aβ1-42-induced hippocampal neurones were treated with AC-YVAD-CMK (caspase-1 inhibitor). Spatial learning and memory ability of mice were detected by morris water maze. Flow cytometry, TUNEL staining, Thioflavin S staining and immunohistochemistry were performed to examine apoptosis and senile plaque deposition. Enzyme linked immunosorbent assay and western blot were performed to assess the levels of protein or cytokines. Co-Immunoprecipitation was performed to verify the interaction between Stargazin and GluA1.ResultsAC-YVAD-CMK treatment improved spatial learning and memory ability and reduced senile plaque deposition of APP/PS1 mice. Moreover, AC-YVAD-CMK promoted membrane transport of GluA1 in APP/PS1 mice. In vitro, Aβ1-42-induced hippocampal neurones exhibited an increase in apoptosis and a decrease in the membrane transport of GluA1, which was abolished by AC-YVAD-CMK treatment. In addition, Stargazin interacted with GluA1, which was repressed by caspase-1. Caspase-1/IL-1β inhibited membrane transport of GluA1 by inhibiting the interaction between Stargazin and GluA1.ConclusionsOur data demonstrate that caspase-1/IL-1β represses membrane transport of GluA1 by inhibiting the interaction between Stargazin in Alzheimer's disease. Thus, caspase-1/IL-1β may be a target for Alzheimer's disease treatment.

Effect of orexin-A on mitochondrial biogenesis, mitophagy and structure in HEK293-APPSWE cell model of Alzheimer's disease.

Mitochondrial dysfunction plays a key role in the pathogenesis and progression of Alzheimer's Disease (AD). Our previous studies showed that over expression of AD-associated mutant β-amyloid precursor protein (APP) led to abnormalities of mitochondrial biogenesis and mitophagy, leading to mitochondrial dysfunction. However, the mechanism remains unclear. In this study, we investigated the effect of orexin-A on mitochondrial biogenesis, mitophagy and mitochondrial structure in overexpression of AD-associated mutant APP cells. We used 20E2 cells as the AD cell model. 20E2 cells were treated with orexin-A (50, 100nmol/L). The effect of different concentrations of orexin-A on cell activity was detected by MTT. As compared with the non-treated 20E2 cells, orexin-A-treated 20E2 cells showed increased expression of APP, decreased cell viability and decreased adenosine triphosphate (ATP) level, decreased levels of regulatory proteins of mitochondrial biogenesis (peroxisome proliferator-activated receptor gamma coactivator 1-alpha [PGC-1α], nuclear respiratory factor 1/2 [NRF1/2], mitochondrial transcription factor A [TFAM]), increased levels of regulatory proteins of mitophagy (Parkin, PTEN-induced putative kinase 1 [PINK1], microtubule-associated protein light chain 3 II/I [LC3-II/LC3-I]) and decreased p62 level, with damaged mitochondrial structure. Orexin-A may reduce mitochondrial biogenesis, enhance mitophagy and damage mitochondrial structure in AD.

MiR-137 overexpression protects neurons from Aβ-induced neurotoxicity via ERK1/2

Accumulation of amyloid-β (Aβ) is implicated in the pathogenesis and development of Alzheimer's disease (AD) and it is a prime suspect for causing neuronal loss and cognitive deficits during AD. To explore the mechanisms of Aβ-induced neurodegeneration, we used Aβ-treated primary cortical neuron culture as a cell model of AD and investigated the function of miR-137 in this process. qRT-PCR and western blot were used to examine levels of miR-137, AMPA receptors (AMPARs), p-ERK1/2, and ERK1/2. MTT assay and caspases 3 activity assay were employed to examine cell viability and apoptosis. Dual-luciferase assay was used to validate the interaction between miR-137 and ERK1/2. We found that Aβ decreased cell viability, AMPAR and miR-137 levels, but increased caspase 3 activity in a dose- and time-dependent manner. Overexpression of miR-137 suppressed the Aβ-induced neurotoxicity. MiR-137 directly bound to ERK1/2 mRNA and negatively regulated its expression. Further, overexpression of ERK1/2 blocked the effects of miR-137 mimics on Aβ-induced neurotoxicity. These results provide strong evidence that miR-137 protects neurons against Aβ-induced neurotoxicity via targeting ERK1/2.

TNF-α-dependent neuronal necroptosis regulated in Alzheimer's disease by coordination of RIPK1-p62 complex with autophagic UVRAG.

Background: Neuronal death is a major hallmark of Alzheimer's disease (AD). Necroptosis, as a programmed necrotic process, is activated in AD. However, what signals and factors initiate necroptosis in AD is largely unknown.Methods: We examined the expression levels of critical molecules in necroptotic signaling pathway by immunohistochemistry (IHC) staining and immunoblotting using brain tissues from AD patients and AD mouse models of APP/PS1 and 5×FAD. We performed brain stereotaxic injection with recombinant TNF-α, anti-TNFR1 neutralizing antibody or AAV-mediated gene expression and knockdown in APP/PS1 mice. For in vitro studies, we used TNF-α combined with zVAD-fmk and Smac mimetic to establish neuronal necroptosis models and utilized pharmacological or molecular biological approaches to study the signaling pathways.Results: We find that activated neuronal necroptosis is dependent on upstream TNF-α/TNFR1 signaling in both neuronal cell cultures and AD mouse models. Upon TNF-α stimulation, accumulated p62 recruits RIPK1 and induces its self-oligomerization, and activates downstream RIPK1/RIPK3/MLKL cascade, leading to neuronal necroptosis. Ectopic accumulation of p62 is caused by impaired autophagy flux, which is mediated by UVRAG downregulation during the TNF-α-promoted necroptosis. Notably, UVRAG overexpression inhibits neuronal necroptosis in cell and mouse models of AD.Conclusions: We identify a finely controlled regulation of neuronal necroptosis in AD by coordinated TNF-α signaling, RIPK1/3 activity and autophagy machinery. Strategies that could fine-tune necroptosis and autophagy may bring in promising therapeutics for AD.

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.

Sirt3 gene knockout protects mice from Alzheimer's disease through activating autophagy

To investigate the protective effect of Sirt3 gene knockout on Alzheimer's disease (AD) in mice. The animal model of AD was established by intraperitoneal injection of D-galactose and brain-localized injection of amyloid β-protein (Aβ)1-40 in wild type C57BL/6 mice and Sirt3 gene knockout mice. Morris water maze, Y maze and tail suspension test were used to assess the cognitive function and anxiety-like behaviors in mice. Aβ deposition in the hippocampus was detected by immunofluorescent staining. Western blotting analysis was conducted to detect the expression of related proteins in the brain. Mouse cortical primary neurons were cultured and AD cell model was established. MTT assay was used to detect cell viability after modeling. Behavioral results showed that cognitive deficits were found in wide type mice after induction of AD as its prolonged escape latency (P<0.05) and decreased crossing number of platform and target zone duration (all P<0.05); while the knockout of Sirt3 alleviated cognitive deficit induced by AD (all P<0.05). Aβ immunofluorescence staining showed that the deposition of Aβ in the hippocampal region and expression of cleaved caspase 3 in the brain in Sirt3 knockout mice was reduced compared with that of wild type mice (all P<0.05). The expression of LC3-Ⅱ and P62 increased after AD was induced in wild type mice, while the autophagy in Sirt3 knockout mice was activated as the increase expression of LC3-Ⅱ and decrease expression of P62 (all P<0.05). In the AD cell model, the results of MTT assay were consistent with the animal experiments, and the protective effect of Sirt3 knockdown was eliminated after the treatment of the autophagy inhibitor chloroquine (all P<0.05). The knockdown of Sirt3 shows a protective effect on AD induced by D-galactose and Aβ1-40 in mice, which may be related to its function of activating autophagy.

Ginsenoside Rb1 protected PC12 cells from Aβ25-35-induced cytotoxicity via PPARγ activation and cholesterol reduction

Accumulating evidences suggest that amyloid β (Aβ)-peptide plays a key role in pathogenesis of Alzheimer's disease (AD) through aggregation and deposition into plaques in neuronal cells. Membrane components such as cholesterol and gangliosides not only enhance the production of amyloidogenic Aβ fragments, but also appear to strengthen Aβ–membrane interaction. Ginsenoside Rb1 (GRb1) is a major active component of Panax, which is widely used to improve learning and memory. In the present study, whether ginsenoside Rb1 could protect pheochromocytoma cells (PC12 cells) from Aβ25-35-induced cytotoxicity including inhibiting cell growth, inducing apoptosis, producing reactive oxygen species (ROS), destroying the cytoskeleton and bringing about membrane toxicity was investigated. Our results indicated that ginsenoside Rb1 could serve as an agonist of peroxisom proliferator-activated receptor-γ (PPARγ) and reduce the level of cholesterol in AD model cells. Reduction of the Aβ25-35-induced cytotoxicity by lowering cholesterol was evidenced by reduction of ROS production, lipid peroxidation, and protection of cytoskeleton and membrane surface rigidity. Most importantly, the viability of PC12 cells increased from 50.42 ± 5.51% for the AD group to 102.72 ± 4.34% for the 50 μM ginsenoside Rb1 group with cholesterol reduction. Our results suggested that ginsenoside Rb1 might function as an effective candidate to promote reverse cholesterol transport and lower ROS production, therefore providing a new insight into prevention and treatment of AD.