Human Neuronal SH-SY5Y Cells Research Articles

Memantine ameliorates oxaliplatin-induced neurotoxicity via mitochondrial protection

ABSTRACT Oxaliplatin is an effective chemotherapeutic agent for the treatment of malignant tumors. However, severe oxaliplatin-induced neurotoxicity has been well documented. Memantine is a drug for the management of Alzheimer’s Disease (AD) due to its promising neuroprotective properties. We hypothesize that Memantine possesses a beneficial role against chemotherapy-induced neuronal damages. In this study, we established an oxaliplatin-induced neurotoxicity assay model in human SHSY-5Y neuronal cells and investigated the protective effect of Memantine. We showed that Memantine treatment ameliorated oxaliplatin-elevated intracellular production of reactive oxygen species (ROS), lipid product malondialdehyde (MDA), and NOX-2 expression. Memantine alleviated impairment of the mitochondrial membrane potential and ATP production by oxaliplatin. As a result, Memantine showed a protective role against oxaliplatin-induced cytotoxicity. Moreover, the terminal deoxynucleotidyl Transferase-mediated dUTP nick end labeling (TUNEL) apoptosis assay revealed that Memantine protected oxaliplatin-induced apoptosis through mitigating the ratio of Bax/Bcl-2 and Caspase-3 cleavage. We concluded Memantine ameliorated the neurotoxicity of oxaliplatin in a mitochondrial-dependent pathway.

Postexercise serum from humans influences the biological tug of war of APP processing in human neuronal cells.

Neurodegenerative diseases such as Alzheimer's disease (AD) are becoming more prevalent in our aging society. One specific neuropathological hallmark of this disease is the accumulation of amyloid-β (Aβ) peptides, which aggregate to form extraneuronal plaques. Increased Aβ peptides are often observed well before symptoms of AD develop, highlighting the importance of targeting Aβ-producing pathways early on in disease progression. Evidence indicates that exercise has the capacity to reduce Aβ peptide production in the brain; however, the mechanisms remain unknown. Exercise-induced signaling mediators could be the driving force behind some of the beneficial effects observed in the brain with exercise. The purpose of this study was to examine if postexercise serum and the factors it contains can alter neuronal amyloid precursor protein (APP) processing. Human SH-SY5Y neuronal cells were differentiated with retinoic acid for 5 days and treated with 10% pre- or postexercise serum from humans for 30 min. Cells were collected for analysis of acute (30 min; n = 6) or adaptive (24 h posttreatment; n = 6) responses. There were no statistical differences in a disintegrin and metalloproteinase 10 (ADAM10) and β-site amyloid precursor protein cleaving enzyme 1 (BACE1) mRNA or protein expression with postexercise serum treatment at either time point. However, there was an increase in the ratio of soluble amyloid precursor protein α (sAPPα) to soluble amyloid precursor protein β (sAPPβ) protein content (P = 0.05) after 30 min of postexercise serum treatment. In addition, 30 min of postexercise serum treatment increased ADAM10 (P = 0.01) and BACE1 (P = 0.02) activity. These findings suggest that postexercise serum modulates important enzymes involved in APP processing, potentially pushing the cascade toward the nonamyloidogenic arm.

Aggregation-prone A53T mutant of α-synuclein exaggerates methamphetamine neurotoxicity in SH-SY5Y cells: Protective role of cellular cholesterol

The aim of this study is to examine the effects of wild type as well as a mutant (A53T) form of α-synuclein (α-syn) on neuronal cells exposed to methamphetamine (METH). SH-SY5Y human dopaminergic neuronal cells stably expressing exogenously added wild type (WT) or A53T α-syn were established for this purpose. Among the three types of cells, parental, WT α-syn-overexpressing, and A53T α-syn overexpressing SH-SY5Y cells (hereafter referred to as SH-SY5Y, WT SH-SY5Y, and A53T SH-SY5Y, respectively), only A53T SH-SY5Y cells showed significant loss of cell viability after exposure to 2 mM METH for 24 h. Transcriptome analysis using DNA microarray showed that METH induced genes for cholesterol biosynthesis in all of these three cell lines, suggesting that METH upregulates cellular cholesterol biosynthesis independently from cellular α-syn levels. Visualization of the cellular localization of free cholesterol showed that METH induces an aberrant intracellular accumulation of free cholesterol in all three cell lines. In addition, we observed the aggregation of α-syn into cytoplasmic granules, which was more apparent with A53T α-syn than WT α-syn, in cells exposed to METH. Furthermore, the cell death observed in METH-treated A53T SH-SY5Y cells was exaggerated by the addition of 2-hydroxypropyl-β-cyclodextrin (CD), a substance used to extract cholesterol from cells. These results suggest that the aggregation of A53T α-syn in METH-treated cells should be involved in cell death. The upregulation of cellular biosynthesis and cholesterol accumulation by METH should play a protective role against A53T α-syn neurotoxicity in METH-treated SH-SY5Y cells.

Ginsenoside F1 Protects the Brain against Amyloid Beta-Induced Toxicity by Regulating IDE and NEP.

Ginsenoside F1, the metabolite of Rg1, is one of the most important constituents of Panax ginseng. Although the effects of ginsenosides on amyloid beta (Aβ) aggregation in the brain are known, the role of ginsenoside F1 remains unclear. Here, we investigated the protective effect of ginsenoside F1 against Aβ aggregation in vivo and in vitro. Treatment with 2.5 μM ginsenoside F1 reduced Aβ-induced cytotoxicity by decreasing Aβ aggregation in mouse neuroblastoma neuro-2a (N2a) and human neuroblastoma SH-SY5Y neuronal cell lines. Western blotting, real-time PCR, and siRNA analysis revealed an increased level of insulin-degrading enzyme (IDE) and neprilysin (NEP). Furthermore, liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis confirmed that ginsenoside F1 could pass the blood–brain barrier within 2 h after administration. Immunostaining results indicate that ginsenoside F1 reduces Aβ plaques in the hippocampus of APPswe/PSEN1dE9 (APP/PS1) double-transgenic Alzheimer’s disease (AD) mice. Consistently, increased levels of IDE and NEP protein and mRNA were observed after the 8-week administration of 10 mg/kg/d ginsenoside F1. These data indicate that ginsenoside F1 is a promising therapeutic candidate for AD.

Extracellular cardiolipin regulates select immune functions of astrocytes in a toll-like receptor (TLR) 4-dependent manner.

Cardiolipin (CL) is a mitochondrial phospholipid that may be released extracellularly from damaged or dying cells where it can act as a signaling molecule by interacting with the surrounding cells. Previous research demonstrates that extracellular CL regulates select immune functions of microglia in a toll-like receptor (TLR) 4-dependent manner. However, the effects of CL on astrocyte functions are unknown. Astrocytes are essential participants in diverse physiological brain functions; however, they become overactivated in chronic neuroinflammatory states, including Alzheimer's disease, contributing to damage and death of surrounding tissues and cells. Since astrocytes express TLR 4, we hypothesized that, by interacting with this receptor, extracellular CL modulates the secretion of cytokines and cytotoxins by astrocytes, as well as their phagocytic activity. We studied the effects of CL added to the culture media of primary murine astrocytes and human U118 MG astrocytic cells, which were used as astrocyte models. Fluorescent latex microspheres and synaptosomes were used to investigate the phagocytic activity of cells. The secretion of inflammatory cytokines was measured by the enzyme-linked immunosorbent assay or immunoblotting. U118 MG astrocyte supernatants were transferred to human SH-SY5Y neuronal cells to assess the secretion of cytotoxins. The expression of TLR 4 and glial fibrillary acidic protein (GFAP) by U118 MG astrocytic cells was quantified by immunostaining and immunoblotting, respectively. Extracellular CL alone upregulated the phagocytic activity of human U118 MG astrocytic cells and their secretion of monocyte chemoattractant protein (MCP)-1 and interferon (IFN)-β. Extracellular CL upregulated the phagocytic activity of primary murine astrocytes. TLR 4-specific antagonist TAK-242 inhibited CL-induced secretion of MCP-1 by human U118 MG cells. CL alone upregulated the cell-surface expression of TLR 4 by the same cell type. Extracellular CL inhibited the lipopolysaccharide (LPS)-induced increase in GFAP expression as well as the secretion of cytotoxins by U118 MG astrocytic cells. Extracellular CL may be used to modulate select functions of astrocytes, which become dysregulated in chronic neuroinflammatory states. Therefore, CL, or liposomes containing this phospholipid, could be used to ameliorate the chronic neuroinflammation and adverse astrocyte activation associated with Alzheimer's disease.

Exposure to PM2.5 induces neurotoxicity, mitochondrial dysfunction, oxidative stress and inflammation in human SH-SY5Y neuronal cells

Ambient air pollution is a global public health issue. Recent evidence suggests that exposure to fine aerosolized particulate matter (PM) as small as ≤2.5 microns (PM2.5) is neurotoxic to brain structures. Many studies also suggest exposure to PM2.5 may cause neurotoxicity and affect brain function. However, the molecular mechanisms by which PM2.5 exerts these effects are not fully understood. Thus, we evaluated the hypothesis that PM2.5 exposure exerts its neurotoxic effects via increased oxidative and inflammatory cellular damage and mitochondrial dysfunction using human SH-SY5Y neuronal cells. Here, we show PM2.5 exposure significantly decreases viability, and increases caspase 3 and 9 protein expression and activity in SH-SY5Y cells. In addition, PM2.5 exposure decreases SH-SY5Y survival, disrupts cell and mitochondrial morphology, and significantly decreases ATP levels, D-loop levels, and mitochondrial mass and function (maximal respiratory function, COX activity, and mitochondrial membrane potential) in SH-SY5Y cells. Moreover, SH-SY5Y cells exposed to PM2.5 have significantly decreased mRNA and protein expression levels of survival genes (CREB and Bcl-2) and neuroprotective genes (PPARγ and AMPK). We further show SH-SY5Y cells exposure to PM2.5 induces significant increases in the levels of oxidative stress, and expression levels of the inflammatory mediator’s TNF-α, IL-1β, and NF-κB. Taken together, these results provide the first evidence of the biochemical, molecular and morphological effects of PM2.5 on human neuronal SH-SY5Y cells, and support our hypothesis that increased mitochondrial disruption, oxidative stress and inflammation are critical mediators of its neurotoxic effects. These findings further improve our understanding of the neuronal cell impact of PM2.5 exposure, and may be useful in the design of strategies for the treatment and prevention of human neurodegenerative disorders.

Homarine Alkyl Ester Derivatives as Promising Acetylcholinesterase Inhibitors.

Reversible acetylcholinesterase (AChE) inhibitors are key therapeutic tools to modulate the cholinergic connectivity compromised in several degenerative pathologies. In this work, four alkyl esters of homarine were synthesized and screened by using Electrophorus electricus AChE and rat brain AChE-rich fraction. Results showed that all homarine alkyl esters are able to inhibit AChE by a competitive inhibition mode. The effectiveness of AChE inhibition increases with the alkyl side chain length of the homarine esters, being HO-C16 (IC50 =7.57±3.32 μM and Ki =18.96±2.28 μM) the most potent inhibitor. The fluorescence quenching studies confirmed that HO-C16 is the compound with higher selectivity and affinity for the tryptophan residues in the catalytic active site of AChE. Preliminary cell viability studies showed that homarine esters display no toxicity for human neuronal SH-SY5Y cells. Thus, the long-chain homarine esters emerge as new anti-cholinesterase agents, with potential to be considered for therapeutic applications development.

The protective effects of Ramelteon against 6-OHDA-induced cellular senescence in human SH-SY5Y neuronal cells.

Background and purposeParkinson's disease (PD) is a severe neurodegenerative disease with high morbidity in the elderly population. 6‐OHDA‐induced cell senescence is reported to be involved in the pathogenesis of PD. Ramelteon is an oral hypnotic agent that specifically targets the receptors of the suprachiasmatic nucleus in the human hypothalamus. Here, an investigation is made to see whether Ramelteon possesses a beneficial effect against 6‐OHDA‐induced cellular senescence in human SH‐SY5Y neuronal cells.MethodsThe release of LDH was detected to assess cytotoxicity and flow cytometry was conducted to evaluate the cell cycle. The telomerase activity and the SA‐β‐Gal assay were performed to determine the state of cell senescence. Oxidative stress was evaluated by detecting the release of H2O2. The expressions of p21, p53, and Nrf2 were measured using the qRT‐PCR and Western blotting assay. siRNA technology was used to knock down the expression level of Nrf2 in SH‐SY5Y neuronal cells.ResultsFirst, it was found that Ramelteon mitigated cell cycle arrest in the G0/G1 phase in 6‐OHDA‐challenged SH‐SY5Y neuronal cells. Second, treatment with Ramelteon alleviated cellular senescence in 6‐OHDA‐treated SH‐SY5Y neuronal cells by increasing telomerase activity and reducing the activity of SA‐β‐gal. It was also found that Ramelteon reduced the expressions of p21 and p53. Notably, Ramelteon attenuated 6‐OHDA‐induced oxidative stress by increasing the expression of Nrf2. Silencing of Nrf2 abolished the protective effects of Ramelteon against 6‐OHDA‐induced cellular senescence. Based on these findings, it was concluded that Ramelteon alleviated 6‐OHDA‐induced cellular senescence by increasing the expression of Nrf2 in human SH‐SY5Y neuronal cells.ConclusionRamelteon protected against 6‐OHDA‐induced cellular senescence in human SH‐SY5Y neuronal cells through activating the Nrf2 signaling pathway.

Organochlorine Pesticide Dieldrin Suppresses Cellular Interferon-Related Antiviral Gene Expression.

Organochlorine pesticides (OCPs) are persistent pollutants linked to diverse adverse health outcomes. Environmental exposure to OCPs has been suggested to negatively impact the immune system but their effects on cellular antiviral responses remain unknown. Transcriptomic analysis of N27 rat dopaminergic neuronal cells unexpectedly detected high level expression of genes in the interferon (IFN)-related antiviral response pathways including the IFN-induced protein with tetratricopeptide repeats 1 and 2 (Ifit1/2) and the MX Dynamin Like GTPases Mx1 and Mx2. Interestingly, treatment of N27 cells with dieldrin markedly downregulated the expression of many of these genes. Dieldrin exterted a similar effect in inhibiting IFIT2 and MX1 gene expression in human SH-SY5Y neuronal cells induced by an RNA viral mimic, polyinosinic: polycytidylic acid (poly I:C) and IFIT2/3 gene expression in human pulmonary epithelial cells exposed to human influenza H1N1 virus. Mechanistically, dieldrin induced a rapid rise in levels of intracellular reactive oxygen species (iROS) and a decrease in intracellular glutathione (GSH) levels in SH-SY5Y cells. Treatment with N-acetylcysteine, an antioxidant and GSH biosynthesis precursor, effectively blocked both dieldrin-induced increases in iROS and its inhibition of poly I:C-induced upregulation of IFIT and MX gene expression, suggesting a role for intracellular oxidative status in dieldrin's modulation of antiviral gene expression. This study demonstrates that dieldrin modulates key genes of the cellular innate immune responses that are normally involved in the host's cellular defense against viral infections. Our findings have potential relevance to understanding the organismal effects of environmentally persistent organochlorine contaminants on the mammalian cellular immune system.

Metabolic profiling in human SH-SY5Y neuronal cells exposed to perfluorooctanoic acid (PFOA)

Perfluorooctanoic acid (PFOA) is an abundant per- and polyfluoroalkyl substance (PFAS) detected in both indoor and outdoor environments. While studies suggest exposure concerns for humans, studies investigating PFOA-induced neurotoxicity are lacking. To address this gap, we exposed differentiated human SH-SY5Y cells to PFOA (0.1 μM up to 500 μM) at different time points (4, 24, 48, and 72 h) and measured cell viability, Casp3/7 activity, ATP levels, ATP synthase enzyme activity, mitochondrial membrane potential, reactive oxygen species (ROS), oxygen consumption rates for mitochondrial stress test (XFe24 Flux analyzer), glucose utilization, and global metabolome profiles to assess the potential for PFOA-induced neurotoxicity. Treatment with 10 or 100 μM PFOA did not compromise cell viability nor induce cytotoxicity to SH-SY5Y cells over a 48-hour exposure period. However, >250 μM PFOA compromised cell viability, induced cytotoxicity, and induced caspase 3/7 activity at 48 h. ATP levels were reduced in cells treated with 400 μM PFOA for 24 and 48 h, and with 100 μM PFOA and higher at 72 h. ATP synthase activity was inhibited by 250 μM PFOA but was unchanged by PFOA treatment at 200 μM or less. Conversely, mitochondrial membrane potential was reduced by >10 μM PFOA after 24 h. Total ROS was increased with 100 μM PFOA and higher after 4 h of exposure. Several mitochondria-related endpoints (basal respiration, ATP production, maximum respiration) were negatively affected at 250 μM PFOA at both 24- and 48-hour exposure, but were unaltered at concentrations of 100 μM PFOA or less. One exception was mitochondrial spare capacity, which was reduced by 100 μM PFOA after 24-hour exposure. Similarly, glycolysis, glycolytic capacity, and glycolytic reserve of SH-SY5Y cells were not altered by 10 nor 100 μM PFOA. Nontargeted metabolomics was conducted in cells treated with either 10 or 100 μM PFOA for 48 h, as these two concentrations were not cytotoxic and 28 metabolites differed among treatments. Notable was that 10 μM PFOA had little effect on the SH-SY5Y metabolome, and the metabolic profile was not statistically different from media nor solvent controls. On the other hand, 100 μM PFOA shifted the metabolic signature of the neuronal cells, leading to reduced abundance of ATP-related metabolites (adenine, nicotinamide), neurotransmitter precursors (DL-tryptophan, l-tyrosine), and metabolites that protect mitochondria during oxidative stress (betaine, orotic acid, and l-acetyl carnitine). We hypothesize that this metabolic signature may be associated with the reduced mitochondrial membrane potential observed at lower PFOA concentrations. Metabolic shifts appear to precede compromised cell viability, cytotoxicity, and apoptosis. This study generates mechanistic knowledge regarding PFOA-induced neurotoxicity, focusing on mitochondrial oxidative respiration and the neuronal metabolome.

Cannabidiol regulates CB1‐pSTAT3 signaling for neurite outgrowth, prolongs lifespan, and improves health span in Caenorhabditis elegans of Aβ pathology models

Cannabidiol (CBD), a phytocannabinoid from the Cannabis sativa plant, exhibits a broad spectrum of potential therapeutic properties for neurodegenerative diseases. An accumulation of amyloid-β (Aβ) protein is one of the most important neuropathology in neurodegenerative diseases like Alzheimer's disease (AD). Data on the effect of CBD on the amelioration of Aβ-induced neurite degeneration and its consequences of life and health spans is sparse. This study aimed to investigate the effects of CBD on neurite outgrowth in cells and lifespan and health span in Caenorhabditis elegans (C. elegans). In human SH-SY5Y neuronal cells, CBD prevented neurite lesion induced by Aβ1-42 and increased the expression of fatty acid amide hydrolase (FAAH) and cannabinoid receptor 1 (CB1R). Furthermore, CBD both protected the reduction of dendritic spine density and rescued the activity of synaptic Ca2+ /calmodulin-dependent protein kinase II (CaMKII) from Aβ1-42 toxicity in primary hippocampal neurons. In C. elegans, we used the transgenic CL2355 strain of C. elegans, which expresses the human Aβ peptide throughout the nervous system and found that CBD treatment extended lifespan and improved health span. The neuroprotective effect of CBD was further explored by observing the dopaminergic neurons using transgenic dat-1: GFP strains using the confocal microscope. This study shows that CBD prevents the neurite degeneration induced by Aβ, by a mechanism involving CB1R activation, and extends lifespan and improves health span in Aβ-overexpressing worms. Our findings support the potential therapeutic approach of CBD for the treatment of AD patients.

Alpha-lipoic acid protects human dopaminergic neuronal cells against hydrogen peroxide-induced cell injury by inhibiting autophagy and apoptosis

Alpha-lipoic acid (ALA) is a naturally occurring antioxidant and has been previously used to treat diabetes and cardiovascular disease. However, the autophagy effects of ALA against oxidative stress-induced dopaminergic neuronal cell injury remain unclear. The aim of this study was to investigate the role of ALA in autophagy and apoptosis against oxidative stress in the SH-SY5Y human dopaminergic neuronal cell line. We examined SH-SY5Y phenotypes using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay (cell viability/proliferation), 4′,6-diamidino-2-phenylindole dihydrochloride nuclear staining, Live/Dead cell assay, cellular reactive oxygen species (ROS) assay, immunoblotting, and immunocytochemistry. Our data showed ALA attenuated hydrogen peroxide (H2O2)-induced ROS generation and cell death. ALA effectively suppressed Bax up-regulation and Bcl-2 and BclxL down-regulation. Furthermore, ALA increased the expression of the antioxidant enzyme, heme oxygenase-1. Moreover, the expression of Beclin-1 and LC-3 autophagy biomarkers was decreased by ALA in our cell model. Combined, these data suggest ALA protects human dopaminergic neuronal cells against H2O2-induced cell injury by inhibiting autophagy and apoptosis.

Superoxide dismutase 1 (SOD1) and cadmium: A three models approach to the comprehension of its neurotoxic effects

Cadmium (Cd) is a widespread toxic environmental contaminant, released by anthropogenic activities. It interferes with essential metal ions homeostasis and affects protein structures and functions by substituting zinc, copper and iron. In this study, the effect of cadmium on SOD1, a CuZn metalloenzyme catalyzing superoxide conversion into hydrogen peroxide, has been investigated in three different biological models. We first evaluated the effects of cadmium combined with copper and/or zinc on the recombinant GST-SOD1, expressed in E. coli BL21. The enzyme activity and expression were investigated in the presence of fixed copper and/or zinc doses with different cadmium concentrations, in the cellular medium. Cadmium caused a dose-dependent reduction in SOD1 activity, while the expression remains constant. Similar results were obtained in the cellular model represented by the human SH-SY5Y neuronal cell line. After cadmium treatment for 24 and 48 h, SOD1 enzymatic activity decreased in a dose- and time-dependent way, while the protein expression remained constant. Finally, a 16 h cadmium treatment caused a 25 % reduction of CuZn-SOD activity without affecting the protein expression in the Caenorhabditis elegans model. Taken together our results show an inhibitory effect of cadmium on SOD1 enzymatic activity, without affecting the protein expression, in all the biological models used, suggesting that cadmium can displace zinc from the enzyme catalytic site.

Viral infection of human neurons triggers strain-specific differences in host neuronal and viral transcriptomes.

Infection with herpes simplex virus 1 (HSV-1) occurs in over half the global population, causing recurrent orofacial and/or genital lesions. Individual strains of HSV-1 demonstrate differences in neurovirulence in vivo, suggesting that viral genetic differences may impact phenotype. Here differentiated SH-SY5Y human neuronal cells were infected with one of three HSV-1 strains known to differ in neurovirulence in vivo. Host and viral RNA were sequenced simultaneously, revealing strain-specific differences in both viral and host transcription in infected neurons. Neuronal morphology and immunofluorescence data highlight the pathological changes in neuronal cytoarchitecture induced by HSV-1 infection, which may reflect host transcriptional changes in pathways associated with adherens junctions, integrin signaling, and others. Comparison of viral protein levels in neurons and epithelial cells demonstrated that a number of differences were neuron-specific, suggesting that strain-to-strain variations in host and virus transcription are cell type-dependent. Together, these data demonstrate the importance of studying virus strain- and cell-type-specific factors that may contribute to neurovirulence in vivo, and highlight the specificity of HSV-1–host interactions.

IL-8 and MCP-1 Impact on Tau Phosphorylation and Phosphatase Activity

Chronic inflammation is a feature of Alzheimer´s disease (AD), resulting in excessive production of inflammatory mediators that can lead to neuroinflammation, contributing to alterations in Aβ production and deposition as Senile Plaques (SPs), and to neurofibrillary tangles (NFTs) formation, due to hyperphosphorylated Tau protein. This work addressed the impact of the interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1), two chemokines, on Tau phosphorylation; and also evaluated the chemokines' levels in plasma using samples from a regional cohort. Human neuronal SH-SY5Y cells exposed to IL-8 and MCP-1 chemokines were monitored for their protein and phosphorylated protein levels by western blotting analysis. A serine/threonine protein phosphatase (PPs) activity assay was employed to monitor PPs activity. Subsequently, flow cytometry was used to monitor chemokines levels in plasma samples from individuals with cognitive deficits. Chemokines' exposure resulted only in minor cytotoxicity effects on SH-SY5Y, and in increased Tau phosphorylation, particularly at the S396 residue. Tau phosphorylation correlated with PPs inhibition and was consistent with GSK3β phosphorylation-mediated inhibition. Subsequent analysis of plasma from individuals with cognitive deficits showed that IL-8 levels were decreased. Data shows that both chemokines tested can exert an effect on GSK3β phosphorylation and modulate PPs activity, potentially resulting in increased Tau phosphorylation and subsequent NFTs formation. One can deduce that increased chemokines stimulation during chronic inflammation can exacerbate this event. The work contributes to a better understanding of the mode of action of these chemokines on AD pathogenesis and opens novel research avenues.

In vitro assessment of the cytotoxic, genotoxic and oxidative stress effects of the synthetic cannabinoid JWH-018 in human SH-SY5Y neuronal cells.

JWH-018 was the first synthetic cannabinoid introduced as a legal high and the first of the new generation of novel psychoactive substances that flooded worldwide drug markets. JWH-018 was marketed as "spice," "herbal incense," or "herbal blend," as a popular and legal (at the time) alternative to cannabis (marijuana). JWH-018 is a potent synthetic cannabinoid with considerable toxicity associated with its use. JWH-018 has qualitatively similar but quantitatively greater pharmacological effects than cannabis, leading to intoxications and even deaths. The mechanisms of action of the drug's toxicity require research, and thus, the aim of the present study was to investigate the toxicological profile of JWH-018 in human SH-SY5Y neuronal cells. SH-SY5Y neuronal cells were exposed to increasing concentrations from 5 to 150μM JWH-018 over 24h. Cytotoxicity, DNA damage, the apoptotic/necrotic rate, and oxidative stress were assessed following SH-SY5Y exposure. JWH-018 did not produce a significant decrease in SH-SY5Y cell viability, did not alter apoptotic/necrotic rate, and did not cause genotoxicity in SH-SY5Y cells with 24-h exposure. Glutathione reductase and catalase activities were significantly reduced; however, there was no significant change in glutathione peroxidase activity. Also, JWH-018 treatment significantly decreased glutathione concentrations, significantly increased protein carbonylation, and significantly increased malondialdehyde (MDA) concentrations. For significance, all P<0.05. JWH-018 produced oxidative stress in SH-SY5Y cells that could be an underlying mechanism of JWH-018 neurotoxicity. Additional in vivo animal and human-based studies are needed to confirm our findings.

Molecular Basis of Preventive Effects of Habitual Coffee Intake against Chronic Diseases

Epidemiological studies have shown that coffee consumption may be associated with a lower risk of developing several chronic disorders. To elucidate the molecular mechanism of the effects of coffee, we analyzed molecular response upon exposure to coffee extract using cellular and animal models of these diseases. As obesity is recognized as a major risk factor for these chronic diseases, we investigated the effect of coffee on adipogenesis using mouse preadipocyte 3T3-L1 cells. We found that coffee induced proteasomal degradation of IRS-1, leading to reduction of PPARγ expression, a master transcription factor for adipogenesis. Reduction in weight as well as in IRS-1 expression was detected in the fat tissues of the high fat-diet-fed mice when reared with 60% coffee for 7 weeks. As for Alzheimer's disease, we analyzed the effect of coffee on amyloid β (Aβ) production in human neuronal SH-SY5Y cells. We found a 20% reduction in Aβ production when treated with 2.5% coffee for 2 d. This reduction was due to proteasomal degradation of BACE1 (β-secretase), which was activated by protein kinase A. In addition, coffee ameliorates LPS-induced inflammatory responses in RAW264.7 macrophages by reducing NFκB activity and Nrf2 activation. Roasted coffee prevents selenite-induced cataractogenesis by ameliorating antioxidant loss. Pyrocatechol, a component of roasted coffee, also reduced Aβ production and exhibits anti-inflammatory effects by a similar mechanism as coffee. Our results suggest that roasting coffee beans to generate pyrocatechol is necessary for the preventive effects of coffee intake on the chronic diseases.

Mesencephalic Astrocyte-Derived Neurotrophic Factor (MANF) Regulates Neurite Outgrowth Through the Activation of Akt/mTOR and Erk/mTOR Signaling Pathways.

Neurite outgrowth is essential for brain development and the recovery of brain injury and neurodegenerative diseases. In this study, we examined the role of the neurotrophic factor MANF in regulating neurite outgrowth. We generated MANF knockout (KO) neuro2a (N2a) cell lines using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 and demonstrated that MANF KO N2a cells failed to grow neurites in response to RA stimulation. Using MANF siRNA, this finding was confirmed in human SH-SY5Y neuronal cell line. Nevertheless, MANF overexpression by adenovirus transduction or addition of MANF into culture media facilitated the growth of longer neurites in RA-treated N2a cells. MANF deficiency resulted in inhibition of Akt, Erk, mTOR, and P70S6, and impaired protein synthesis. MANF overexpression on the other hand facilitated the growth of longer neurites by activating Akt, Erk, mTOR, and P70S6. Pharmacological blockade of Akt, Erk or mTOR eliminated the promoting effect of MANF on neurite outgrowth. These findings suggest that MANF positively regulated neurite outgrowth by activating Akt/mTOR and Erk/mTOR signaling pathways.

Mitochondrial DNA Manipulations Affect Tau Oligomerization.

Mitochondrial dysfunction and tau aggregation occur in Alzheimer's disease (AD), and exposing cells or rodents to mitochondrial toxins alters their tau. To further explore how mitochondria influence tau, we measured tau oligomer levels in human neuronal SH-SY5Y cells with different mitochondrial DNA (mtDNA) manipulations. Specifically, we analyzed cells undergoing ethidium bromide-induced acute mtDNA depletion, ρ0 cells with chronic mtDNA depletion, and cytoplasmic hybrid (cybrid) cell lines containing mtDNA from AD subjects. We found cytochrome oxidase activity was particularly sensitive to acute mtDNA depletion, evidence of metabolic re-programming in the ρ0 cells, and a relatively reduced mtDNA content in cybrids generated through AD subject mitochondrial transfer. In each case tau oligomer levels increased, and acutely depleted and AD cybrid cells also showed a monomer to oligomer shift. We conclude a cell's mtDNA affects tau oligomerization. Overlapping tau changes across three mtDNA-manipulated models establishes the reproducibility of the phenomenon, and its presence in AD cybrids supports its AD-relevance.

Neuroactivity of naturally occurring proaporphine alkaloid, pronuciferine.

Pronuciferine is a naturally occurring proaporphine alkaloid that belongs to isoquinoline alkaloids. The aim of this study is to investigate the neuroactivity of pronuciferine. We assessed the neuroprotective effect of pronuciferine against hydrogen peroxide (H2 O2 )-induced apoptosis in human neuronal SH-SY5Y cells. In addition, we measured the effect of pronuciferine on cell metabolites and brain-derived neurotrophic factor (BDNF) level in SH-SY5Y cells. In vitro result shows that pronuciferine at 10 µM significantly (P < .001) increased the proliferation of SH-SY5Y by 45%, and upon H2 O2 addition, pronuciferine significantly (P < .001) suppressed neuronal death caused by H2 O2 . Gas Chromatography-Mass Spectrometry (GC-MS) metabolomics study revealed that pronuciferine has a high impact on glycine-serine-threonine pathway by changing the intracellular level of serine dimethylglycine, sarcosine, and threonine. Also, pronuciferine increased the intercellular level of aspartic acid, glutamine, and tryptophan. Additionally, pronuciferine significantly (P < .05) increased the intracellular BDNF protein expression at 10 µM. Therefore, pronuciferine is a neuroactive molecule that might act as a neuroprotective agent to prevent apoptosis in neurodegenerative diseases.