Human Neuroblastoma Cell Line SH-SY5Y Research Articles

Lichen Based Synthesis of Zinc Oxide Nanoparticles and Evaluation of its Neurotoxic Effects on Human Neuroblastoma Cells

Nanoparticles, smaller than 100 nm are synthesized by chemical and physical methods. Biological synthesis of nanoparticles is very popular in science recently. The aim of the study is green synthesis of zinc oxide nanoparticles (ZnO NPs) using the lichen extract (Ramalina fraxinea) and investigating the cytotoxic effects of ZnO NPs on human neuroblastoma cells (SHSY-5Y). Despite the widespread use of ZnO NPs, a limited number of studies have investigated the neurobiological effects of ZnO NP. Therefore, we tested the neurotoxic effect of green synthesized ZnO NPs administration and its neuroprotective effect against hydrogen peroxide-induced cell damage on SH-SY5Y human neuroblastoma cell line. The absorbance peak of the ZnO NPs was detected by UV–visible spectroscopy (UV-Vis) at 330 nm. The average diameter of ZnO NPs was measured as about 21 nm by Scanning Electron Microscope (SEM) and Field Emission Scanning Electron Microscope (FE-SEM) images. According to X-ray Diffraction (XRD) diagram, ZnO NPs were hexagonal in structure. The peaks observed in the Fourier Transform Infrared (FT-IR) test showed functional groups in the structure of the nanoparticles. According to our results, ZnO NPs may have beneficial effects at the low concentrations while neurotoxic effects at the higher doses in SH-SY5Y. In addition, we indicate that hydrogen peroxide-induced cell death could not reverse by ZnO NPs and its higher doses potentiated the neurodegenerative effect of hydrogen peroxide. In conclusion, here we report that ZnO NPs, widely used in various products, may have beneficial or harmful effects in a dose-dependent manner and play a role in neuropsychiatric disease, especially neurodegenerative diseases. This is the first study dealing with neurotoxicity on SHSY-5Y of Ramalina fraxinea extract based ZnO NPs.

Long noncoding RNA NORAD regulates MPP+-induced Parkinson’s disease model cells

BackgroundLong non-coding RNAs (lncRNAs) have been demonstrated to play important roles in human diseases. Yet, the functions of lncRNAs in neurodegenerative disorders, such as Parkinson's disease (PD) are poorly understood. In this study, we used human neuroblastoma SH-SY5Y cell line as a cell-basedin vitro PD model, and investigated the role of lncRNA, Non-Coding RNA Activated By DNA Damage (NORAD) in 1-methyl-4-phenylpyridinium (MPP+)-induced PD-like cytotoxicity. MethodsSH-SY5Y cells were culturedin vitro, and treated with MPP + at various concentrations, or of various durations of times to induce PD-like cytotoxic events. qRT-PCR was used to measure MPP+-induced NORAD expression changes. Lentiviral transduction was applied to stably upregulate or downregulate NORAD in SH-SY5Y cells. The effects of NORAD upregulation or downregulation on MPP+-induced cytotoxic events, including dose-dependent and time-dependent cell death, apoptosis, caspase 3/7, reactive Oxygen Species (ROS) and lactate dehydrogenase (LDH) activities, were quantitatively investigated. ResultsMPP + induced cytotoxicity, and downregulated NORAD in both dose- and time- dependent manners in SH-SY5Y cells. Lentiviral-induced NORAD upregulation was found to protect against MPP+-induced cytotoxicity in SH-SY5Y cells, as it rescued MPP+-induced cellular destruction and apoptosis, as well as decreased MPP+-induced caspase 3/7, ROS and LDH activities. Alternatively, NORAD downregulation was found to significantly deteriorate MPP+-induced cytotoxicity in SH-SY5Y cells. ConclusionWe presented a novel functional role of lncRNA NORAD in regulating human Parkinson’s disease.

Extraction of phospholipid-rich fractions from egg yolk and development of liposomes entrapping a dietary polyphenol with neuroactive potential

A new protocol to obtain egg yolk phospholipids in ethanol is presented. Rutin-phospholipids nanoliposomes were prepared and characterized. The procedure takes advantage of the different solubility of egg yolk lipids in ethanol and acetone at low temperature, to efficiently obtain a phospholipid-rich fraction of high purity degree. The phospholipid content in the final fraction is 208.65 ± 26.46 μmol/g fresh egg yolk (16%), accounting for ca. 96% of the extract's dry weight. The phospholipid-rich fraction contains cholesterol (0.069–0.082 cholesterol/phospholipid molar ratio), and vestigial amounts of lutein and zeaxanthin (89.24 ± 9.76 and 14.9 ± 2.16 ng/g of fresh egg yolk, respectively). Saturated fatty acids dominate the extracted phospholipids (50% of egg's total yolk phospholipids), the levels of monounsaturated ranging from 20 to 25%, and polyunsaturated up to 35%. Rutin-liposomes, prepared with phospholipid-rich fraction, presented mean diameter <140 nm, negative surface charge (Zeta potential ~ −13 mV), and entrapment efficiency of rutin up to 87%. In human neuroblastoma cell line SH-SY5Y, rutin-liposomes (lipid 25 μM + rutin 16.7 μM) attenuated glutamate-induced cytotoxicity, in part by reducing the formation of intracellular reactive species, pointing to their potential application as new functional neuroprotective agents.

Tanshinone I Induces Mitochondrial Protection by a Mechanism Involving the Nrf2/GSH Axis in the Human Neuroblastoma SH-SY5Y Cells Exposed to Methylglyoxal.

Methylglyoxal (MG) is a dicarbonyl molecule exhibiting high reactivity and is a major responsible for glycation in human cells. Accumulation of MG is seen in certain diseases, including metabolic disturbances and neurodegeneration. Among other effects, MG promotes mitochondrial dysfunction, leading to bioenergetic decline and redox impairment in virtually any nucleated human cells. The detoxification of MG is dependent on the availability of reduced glutathione (GSH), a major antioxidant that is also utilized in phase II detoxification reactions. The synthesis of GSH is mainly controlled by the transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2). The activation of Nrf2 is stimulated by several reactive compounds, including natural molecules produced by plants. Tanshinone I (T-I) is obtained from Salvia miltiorrhiza Bunge and exerts potent cytoprotective actions in different cell types. Thus, we have investigated here whether and how T-I would be able to protect mitochondria of the human neuroblastoma SH-SY5Y cell line exposed to MG. The cells were pretreated with T-I at 2.5μM for 2h before the challenge with MG at 500μM. T-I significantly attenuated the MG-induced loss of cell viability, bioenergetic decline, and redox impairment in SH-SY5Y cells. The inhibition of the GSH synthesis by buthionine sulfoximine (BSO) at 100μM suppressed the mitochondrial protection promoted by T-I. The silencing of Nrf2 by small interfering RNA (siRNA) abrogated the synthesis of GSH and the mitochondrial protection stimulated by T-I in SH-SY5Y cells. Therefore, T-I induced mitochondrial protection by a mechanism involving the Nrf2/GSH axis in MG-challenged SH-SY5Y cells.

Phenolic constituents from the fresh pericarps of Juglans sigillata

Three new phenolic compounds, 2,3,4-trihydroxy-1-(4′-hydroxybenzyl)benzoic acid (1), 2,3,4-trihydroxy-1-(4′-hydroxy-3′-methoxybenzyl)benzoic acid (2), and 2-(4′-hydroxy-3′-methoxybenzyl)-3,5-dimethoxy-1,4-benzoquinone (3), were isolated from the fresh pericarps of Juglans sigillata Dode. Their structures were elucidated by integrated spectroscopic techniques. Bioactivity screening results showed that compounds 1–3 exhibited moderate neuroprotective effects against H2O2-induced or CoCl2-induced cellular damage in human neuroblastoma SH-SY5Y cell line.

Bioorganic investigation of encapsulated Cysteine derivative into polymeric nanocarrier

In this work, the copolymer-based synthesized Cysteine-loaded nanocarriers prepared by a routine protocol, coprecipitation method. It is the first report to investigate the neuroprotective potential and biocompatibility of Cysteine derivatives loaded into poly(ethylene glycol)-block-poly(e−caprolactone) methyl ether (PEG-b-PCL). The average size of the polymeric/empty NCs was 89 nm and for polymeric/Synthesized derivative of Cysteine was 126 nm. The Drug Loading efficiency was 81%. The concentration of Polymeric NCs was 2.1 x 10 10 particles/ml and the zeta potential of polymeric/empty and polymeric/ Synthesized derivative of Cysteine NCs -5 mV and -11 mV respectively. Biological part of this work were investigated in the SH-SY5Y human neuroblastoma cell line using cell viability and toxicity assays. The concentration of polymeric NCs below 1 x 10 10 particles/ml described as a zero-point damageable for the cell line. Also the Synthesized derivative of Cysteine encapsulated into polymeric NCs have more neuroprotective effect compared to free Cysteine at lower concentration, and therefore, have a significant neuroprotective potential against Z-VAD-fmk and St-evoked SH-SY5Y cell damage.

1-(2',5'-Dihydroxyphenyl)-3-(2-fluoro-4-hydroxyphenyl)-1-propanone (RGM079): A Positive Allosteric Modulator of α7 Nicotinic Receptors with Analgesic and Neuroprotective Activity.

Acetylcholine α7 nicotinic receptors are widely expressed in the brain, where they are involved in the central processing of pain as well as in neuropsychiatric, neurodegenerative, and inflammatory processes. Positive allosteric modulators (PAMs) show the advantage of allowing the selective regulation of different subtypes of acetylcholine receptors without directly interacting with the agonist binding site. Here, we report the preparation and biological activity of a fluoro-containing compound, 1-(2',5'-dihydroxyphenyl)-3-(2-fluoro-4-hydroxyphenyl)-1-propanone (8, RGM079), that behaves as a potent PAM of the α7 receptors and has a balanced pharmacokinetic profile and antioxidant properties comparable or even higher than well-known natural polyphenols. In addition, compound RGM079 shows neuroprotective properties in Alzheimer's disease (AD)-toxicity related models. Thus, it causes a concentration-dependent neuroprotective effect against the toxicity induced by okadaic acid (OA) in the human neuroblastoma cell line SH-SY5Y. Similarly, in primary cultures of rat cortical neurons, RGM079 is able to restore the cellular viability after exposure to OA and amyloid peptide Aβ1-42, with cell death almost completely prevented at 10 and 30 μM, respectively. Finally, compound RGM079 shows in vivo analgesic activity in the complete Freund's adjuvant (CFA)-induced paw inflammation model after intraperitoneal administration.

Transcriptomics and proteomics analysis of Aβ (1-42)-induced neurotoxicity

Backgrounds: β-Amyloid (Aβ) is a principal constituent of senile plaques in Alzheimer’s disease (AD) and induces neuronal cell death. The molecular mechanism of how Aβ evokes neuronal cell death remains complicated, which were investigated in the present study. Methods: Using the human neuroblastoma cell line SHSY5Y, we investigated the neurotoxic effects of human β-Amyloid 1–42 (Aβ1–42) aggregates on gene expression profile and protein expression profile by using the Agilent GeneChip Human 1A (V2) Oligo MicroArray, Quantitative Real-time PCR, PF-2D and Western blot analysis. Results: Our results show that Aβ1–42 specifically influences gene and protein expression such as EGR1, eIF5A, PDE8A, ERp57 and ERp5 in pathways associated with apoptotic process, protein translation, cAMP catabolic process and response to endoplasmic reticulum stress. Conclusion: Although Genes with significant changes in transcriptomic analysis matched very few of the proteins identified in proteomics analysis, our findings will strengthen our knowledge concerning the molecular mechanisms underlying AD.

Orexin-A protects against oxygen-glucose deprivation/reoxygenation-induced cell damage by inhibiting endoplasmic reticulum stress-mediated apoptosis via the Gi and PI3K signaling pathways

The neuropeptide orexin-A (OXA) has a neuroprotective effect, acting as an anti-apoptotic factor in response to multiple stimuli. Apoptosis induced by endoplasmic reticulum stress (ERS) underlies oxygen-glucose deprivation and reoxygenation (OGD/R)-induced cell damage, an in vitro model of ischemia/reperfusion injury. However, that OXA inhibits ERS-induced apoptosis in the OGD/R model has not been reported. In the present study, we investigated the neuroprotective effect of OXA (0.1 μM) on OGD/R-induced damage in the human neuroblastoma cell line SH-SY5Y. After OXA treatment following 4 h oxygen-glucose deprivation (OGD) and then 4 h reoxygenation (R), cell morphology, viability, and apoptosis were analyzed by histology, Cell Counting Kit-8 assay, and flow cytometry, respectively. Western blotting was used to measure expression levels of ERS- and apoptosis-related proteins. To determine signaling pathways involved in OXA-mediated neuroprotection, the Gi pathway inhibitor pertussis toxin (PTX; 100 ng/mL) and PI3K inhibitor LY294002 (LY; 10 μM) were added. In addition, in order to prove the specificity of these characteristics, the OXA antagonist Suvorexant (DORA; Ki of 0.55 nM and 0.35 nM for OX1R and OX2R) was used for intervention. Our results showed that OGD/R induced cell damage, manifested as morphological changes and a significant decrease in viability. Furthermore, Western blotting detected an increase in ERS-related proteins GRP78, p-IRE1α, p-JNK, and Cleaved caspase-12, as well as apoptosis-related proteins Cleaved caspase-3 and Bax, and a decrease in the anti-apoptosis factor Bcl-2. OXA intervention alleviated the degree of cellular damage, and protein expression was also reversed. In addition, the protective effect of OXA was reduced by adding PTX and LY. Meanwhile, after the use of DORA, changes in the expression of related proteins were detected, and it was found that the protective effect of OXA was weakened. Collectively, our results indicate that OXA has a neuroprotective effect on OGD/R-induced cell damage by inhibiting ERS-induced apoptosis through the combined action of Gi and PI3K signaling pathways. These findings help to clarify the mechanism underlying the neuroprotective action of OXA, which should aid the development of further candidate drugs, and provide a new therapeutic direction for the treatment of ischemic stroke.

NEAT1 regulates MPP+-induced neuronal injury by targeting miR-124 in neuroblastoma cells

Long non-coding RNAs (lncRNAs) have been reported to play important roles in Parkinson’s disease (PD) pathogenesis. It was indicated that lncRNA nuclear enriched abundant transcript 1 (NEAT1) is involved in PD. However, the underlying mechanism of NEAT1 is still not fully explored. Human neuroblastoma cell line SH-SY5Y was treated with 1-Methyl-4-phenylpyridinium (MPP+) to mimic PD model in vitro. qRT-PCR was employed to detect the expression of NEAT1, IL-1β, IL-6 and TNF-α. Starbase database, RNA pull-down assay and RNA immunoprecipitation (RIP) assay were performed to verify the relationship between NEAT1 and miR-124. MTT assay and flow cytometry assay were used to detect cell viability and apoptosis. Elisa was introduced to measure the levels of IL-1β, IL6 and TNF-α in culture media. We found that NEAT1 expression was significantly increased in SH-SY5Y cells after MPP+ treatment in dose- and time- dependent manners. MPP+ induced the expression and secretion of IL-1β, IL-6 and TNF-α, inhibited cell viability and induced apoptosis while these effects could be rescued by NEAT1 silencing. miR-124 was a target of NEAT1. Anti-miR-124 could reverse the effects caused by NEAT1 knockdown in MPP+ treated SH-SY5Y cells. Therefore, we speculated that NEAT1 may regulate MPP+ induced neuronal injury by targeting miR-124 in SH-SY5Y cells.

Synthesis and biological evaluation of indoloquinoline alkaloid cryptolepine and its bromo-derivative as dual cholinesterase inhibitors

Alkaloids have always been a great source of cholinesterase inhibitors. Numerous studies have shown that inhibiting acetylcholinesterase as well as butyrylcholinetserase is advantageous, and have better chances of success in preclinical/ clinical settings. With the objective to discover dual cholinesterase inhibitors, herein we report synthesis and biological evaluation of indoloquinoline alkaloid cryptolepine (1) and its bromo-derivative 2. Our study has shown that cryptolepine (1) and its 2-bromo-derivative 2 are dual inhibitors of acetylcholinesterase and butyrylcholinesterase, the enzymes which are involved in blocking the process of neurotransmission. Cryptolepine inhibits Electrophorus electricus acetylcholinesterase, recombinant human acetylcholinesterase and equine serum butyrylcholinesterase with IC50 values of 267, 485 and 699 nM, respectively. The 2-bromo-derivative of cryptolepine also showed inhibition of these enzymes, with IC50 values of 415, 868 and 770 nM, respectively. The kinetic studies revealed that cryptolepine inhibits human acetylcholinesterase in a non-competitive manner, with ki value of 0.88 µM. Additionally, these alkaloids were also tested against two other important pathological events of Alzheimer’s disease viz. stopping the formation of toxic amyloid-β oligomers (via inhibition of BACE-1), and increasing the amyloid-β clearance (via P-gp induction). Cryptolepine displayed potent P-gp induction activity at 100 nM, in P-gp overexpressing adenocarcinoma LS-180 cells and excellent toxicity window in LS-180 as well as in human neuroblastoma SH-SY5Y cell line. The molecular modeling studies with AChE and BChE have shown that both alkaloids were tightly packed inside the active site gorge (site 1) via multiple π-π and cation-π interactions. Both inhibitors have shown interaction with the allosteric “peripheral anionic site” via hydrophobic interactions. The ADME properties including the BBB permeability were computed for these alkaloids, and were found within the acceptable range.

An Efficient Automated Radiosynthesis and Bioactivity Confirmation of VMAT2 Tracer [18F]FP-(+)-DTBZ.

The aim of this study was to optimize the radiolabeling method of [18F]fluoropropyl-(+)-dihydrotetrabenazine ([18F]FP-(+)-DTBZ) to fulfill the demand of preclinical and clinical application. Optimized labeling conditions were performed by altering the molar ratio of precursor to base (P/B), base species, solvents, reaction temperature, reaction time, and precursor concentration through manual radiosynthesis of [18F]FP-(+)-DTBZ. The conditions with the highest radiochemical yield (RCY) were applied to automated radiosynthesis, and the crude product was purified with a Sep-Pak Plus C18 cartridge. Quality control and stability of [18F]FP-(+)-DTBZ were carried out by HPLC. In vitro cellular uptake and blocking assays were conducted in human neuroblastoma cell line SH-SY5Y. In vivo imaging with small animal positron emission tomography (microPET) was performed with Sprague-Dawley rats. Under the optimized conditions (P/K2CO3 = 1:8, heating at 120°C for 3min in dimethyl sulfoxide), an RCY of 88.7% was obtained with 1.0mg precursor. The optimized reaction conditions were successfully applied to an automated module and gave a high activity yield (AY) of 30-55% in about 40min with a > 99.0% radiochemical purity (RCP) and a > 44.4GBq/μmol molar activity (Am). Stability test displayed that the RCP retained > 98.0% in 8h in saline and in phosphate buffer saline (PBS, pH7.4). In vitro cellular uptake assay showed accumulation of [18F]FP-(+)-DTBZ in SH-SY5Y cells, which could be significantly inhibited by vesicular monoamine transporter 2 (VMAT2) inhibitor DTBZ. MicroPET images of rat brain displayed that the striatum showed the highest uptake with a standardized uptake value (SUV) of 3.91 ± 0.30 at ~ 70min. Co-injection with DTBZ (1.0mg/kg) resulted in a 75% decrease of the striatal SUV, confirming the specificity of [18F]FP-(+)-DTBZ to VMAT2. We obtained an optimized radiolabeling method of [18F]FP-(+)-DTBZ and successfully applied it to a commercial available module. The automated synthesis gave a high AY and RCP of [18F]FP-(+)-DTBZ with high and specific binding to VMAT2, facilitating its routine application for VMAT2 tracing.

Protective effect of gastrodin against methamphetamine-induced autophagy in human dopaminergic neuroblastoma SH-SY5Y cells via the AKT/mTOR signaling pathway

Methamphetamine (METH) has been shown to induce neuropathological dysfunction and irreversible brain cell damage. Prior studies indicated the involvement of autophagy in METH-induced neurotoxicity. However, the underlying mechanism by which autophagy contributes to METH-induced neurotoxicity remains elusive. Gastrodin, a primary bioactive constituent of Gastrodia elata—an orchid used in traditional Chinese medicine—is used widely to treat stroke, dementia, and headache. This study investigates whether METH induces autophagy in the human dopaminergic neuroblastoma cell line SH-SY5Y, then examines the neuroprotective effects of gastrodin against autophagy in METH-treated SH-SY5Y cells. The effects of METH on the protein expressions of autophagy-related genes (LC3B and Beclin-1) were evaluated with and without gastrodin. The presence of autophagosomes in the METH-induced treatment with and without gastrodin is revealed through transmission electron microscopy. Pharmacological intervention was employed to study the role of the AKT/mTOR signaling pathway in the gastrodin-mediated neuroprotection against METH-induced autophagy. The present results indicate that METH exposure elevates the protein expression levels of LC3B and Beclin-1 in a dose- and time-dependent manner. Gastrodin is observed to block the METH-induced upregulation of LC3B and Beclin-1 protein expression significantly. Gastrodin is found to exhibit an anti-autophagic effect on the inhibition of the METH-induced Beclin-1 protein expression, partly via the AKT/mTOR These findings may aid the development of a gastrodin-based therapeutic strategy for treating METH-induced neurotoxicity.

Cellular and sub-cellular Cu isotope fractionation in the human neuroblastoma SH-SY5Y cell line: proliferating versus neuron-like cells.

Cu isotope fractionation was investigated in the human neuroblastoma SH-SY5Y cell line, in a proliferating/tumor phase (undifferentiated cells), and in a differentiated state (neuron-like cells), induced using retinoic acid (RA). The SH-SY5Y cell line displays genetic aberrations due to its cancerous origin, but differentiation drives the cell line towards phenotypes suitable for the research of neurological diseases (e.g., Alzheimer's disease or Parkinson's disease). Cellular Cu distribution was first explored by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) imaging and, subsequently, Cu isotopic analysis was performed at cellular and sub-cellular levels via multi-collector inductively coupled plasma-mass spectrometry (MC-ICP-MS). The SH-SY5Y cells showed a re-distribution of intracellular Cu upon RA differentiation. Both undifferentiated and differentiated cells became systematically enriched in the light 63Cu isotope with increasing intracellular Cu content. Differentiated neuron-like SH-SY5Y cells showed a heavier Cu isotopic composition (+ 0.3‰) than did the undifferentiated proliferating cells when exposed to Cu for 24h. However, after a longer exposure time (72h), no difference was observed between both cellular phenotypes. Mitochondrial fractions were enriched in the light 63Cu isotope, compared to whole cells, for both undifferentiated and differentiated cells (no significant difference). The Cu isotopic composition of the remaining cell lysates was heavier than that of the whole cells and + 0.2‰ heavier in the differentiated cells than in the undifferentiated cells. These results indicate that neuronal differentiation affects the Cu isotope fractionation accompanying Cu uptake in the cells, but this effect does not seem to be associated with the mitochondrial Cu pathway. Cu isotope fractionation can be an interesting tool for studying Cu metabolism at a (sub)-cellular level in functional neurons. Graphical abstract.

Site-specific glycation of Aβ1–42 affects fibril formation and is neurotoxic

Aβ1-42 is involved in Alzheimer's disease (AD) pathogenesis and is prone to glycation, an irreversible process where proteins accumulate advanced glycated end products (AGEs). Nϵ-(Carboxyethyl)lysine (CEL) is a common AGE associated with AD patients and occurs at either Lys-16 or Lys-28 of Aβ1-42. Methyglyoxal is commonly used for the unspecific glycation of Aβ1-42, which results in a complex mixture of AGE-modified peptides and makes interpretation of a causative AGE at a specific amino acid residue difficult. We address this issue by chemically synthesizing defined CEL modifications on Aβ1-42 at Lys-16 (Aβ-CEL16), Lys-28 (Aβ-CEL28), and Lys-16 and -28 (Aβ-CEL16&28). We demonstrated that double-CEL glycations at Lys-16 and Lys-28 of Aβ1-42 had the most profound impact on the ability to form amyloid fibrils. In silico predictions indicated that Aβ-CEL16&28 had a substantial decrease in free energy change, which contributes to fibril destabilization, and a increased aggregation rate. Single-CEL glycations at Lys-28 of Aβ1-42 had the least impact on fibril formation, whereas CEL glycations at Lys-16 of Aβ1-42 delayed fibril formation. We also tested these peptides for neuronal toxicity and mitochondrial function on a retinoic acid-differentiated SH-SY5Y human neuroblastoma cell line (RA-differentiated SH-SY5Y). Only Aβ-CEL16 and Aβ-CEL28 were neurotoxic, possibly through a nonmitochondrial pathway, whereas Aβ-CEL16&28 showed no neurotoxicity. Interestingly, Aβ-CEL16&28 had depolarized the mitochondrial membrane potential, whereas Aβ-CEL16 had increased mitochondrial respiration at complex II. These results may indicate mitophagy or an alternate route of metabolism, respectively. Therefore, our results provides insight into potential therapeutic approaches against neurotoxic CEL-glycated Aβ1-42.

Cholinergic Differentiation of Human Neuroblastoma SH-SY5Y Cell Line and Its Potential Use as an In vitro Model for Alzheimer's Disease Studies.

Cholinergic transmission is critical to high-order brain functions such as memory, learning, and attention. Alzheimer's disease (AD) is characterized by cognitive decline associated with a specific degeneration of cholinergic neurons. No effective treatment to prevent or reverse the symptoms is known. Part of this might be due to the lack of in vitro models that effectively mimic the relevant features of AD. Here, we describe the characterization of an AD in vitro model using the SH-SY5Y cell line. Exponentially growing cells were maintained in DMEM/F12 medium and differentiation was triggered by the combination of retinoic acid (RA) and BDNF. Both acetylcholinesterase (AChE) and choline acetyltransferase (ChAT) enzymatic activities and immunocontent were determined. For mimicking tau and amyloid-β pathology, RA + BDNF-differentiated cells were challenged with okadaic acid (OA) or soluble oligomers of amyloid-β (AβOs) and neurotoxicity was evaluated. RA + BDNF-induced differentiation resulted in remarkable neuronal morphology alterations characterized by increased neurite density. Enhanced expression and enzymatic activities of cholinergic markers were observed compared to RA-differentiation only. Combination of sublethal doses of AβOs and OA resulted in decreased neurite densities, an in vitro marker of synaptopathy. Challenging RA + BDNF-differentiated SH-SY5Y cells with the combination of sublethal doses of OA and AβO, without causing considerable decrease of cell viability, provides an in vitro model which mimics the early-stage pathophysiology of cholinergic neurons affected by AD.

Carnosic Acid Pretreatment Attenuates Mitochondrial Dysfunction in SH-SY5Y Cells in an Experimental Model of Glutamate-Induced Excitotoxicity.

Mitochondria are the major site of adenosine triphosphate (ATP) production in mammalian cells. Moreover, mitochondria produce most of the reactive oxygen species (ROS) in nucleated cells. Redox and bioenergetic abnormalities have been seen in mitochondria during the onset and progression of neurodegenerative diseases. In that context, excitotoxicity induced by glutamate (GLU) plays an important role in mediating neurotoxicity. Several drugs have been used in the treatment of diseases involving excitotoxicity. Nonetheless, some patients (20-30%) present drug resistance. Thus, it is necessary to find chemicals able to attenuate mitochondrial dysfunction in the case of excitotoxicity. In this work, we treated the human neuroblastoma SH-SY5Y cell line with the diterpene carnosic acid (CA) at 1μM for 12h prior to the exposure to GLU for further 24h. We found that CA prevented the GLU-induced mitochondrion-related redox impairment and bioenergetic decline in SH-SY5Y cells. CA also downregulated the pro-apoptotic stimulus elicited by GLU in this experimental model. CA exerted mitochondrial protection by a mechanism associated with the transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2), since silencing of this protein with small interfering RNA (siRNA) suppressed the CA-induced protective effects. Future directions include investigating whether CA would be able to modulate mitochondrial function and/or dynamics in in vivo experimental models of excitotoxicity.

Treatment of human neuroblastoma cell line SH-SY5Y with HSP27 siRNA tagged-exosomes decreased differentiation rate into mature neurons.

Heat shock proteins (HSPs) participate in the regulation of different cell activities in response to stimuli. By applying different strategies, the modulation of heat shock proteins is at the center of attention. Conventional delivery approaches are not fully encouraged due to cytotoxicity and immunogenicity issues. Exosomes are touted as bio-shuttles for delivery of distinct biomolecules inside the cells. Here, we aimed to HSP27 small interfering RNA (siRNA)-tagged exosomes for the inhibition of Hsp27 in human neuroblastoma cell line SH-SY5Y and explored differentiation into neuron-like cells. Exosomes were isolated, characterized by scanning electron microscope (SEM) and CD63 then enriched with siRNA against Hsp27. Neuroblastoma cells were incubated with exosomes carrying siRNA for 48 hr. Exosome uptake was monitored by immunofluorescence assay. The cell viability and proliferation were analyzed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and bromodeoxyuridine/5-bromo-2'-deoxyuridine incorporation assays. The ability of cells to form colonies was evaluated by clonogenic assay. The cell potential to express NeuN, a mature neuron factor, was studied by flow cytometry analysis. SEM showed the nano-sized particles and a high level of CD63 after enrichment. Immunofluorescence imaging revealed an appropriate transfection rate in cell exposed to Hsp27 siRNA tagged exosomes. The cell viability and proliferation were reduced compared to cells received nude exosomes ( p < 0.05). Clonogenic activity of cells was diminished by the inhibition of Hsp27. Flow cytometry analysis revealed that the inhibition of Hsp27 prohibited NeuN content, showing the maturation of SH-SY5Y cells to mature cells compared to control. These data confirmed that exosomes could be used as appropriate bio-shuttles for the inhibition of Hsp27-aborted cell differentiation toward mature neuron.

MicroRNA-124 regulates the expression of p62/p38 and promotes autophagy in the inflammatory pathogenesis of Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by motor and nonmotor symptoms due to the selective loss of midbrain dopaminergic neurons. The evidence for a chronic inflammatory reaction mediated by microglial cells in the brain is particularly strong in PD. In our previous study, we have shown that brain-specific microRNA-124 (miR-124) is significantly down-regulated in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of PD and that it can also inhibit neuroinflammation during the development of PD. However, further investigation is required to understand whether the abnormal expression of miR-124 regulates microglial activation. In this study, we found that the expression of sequestosome 1 (p62) and phospho-p38 mitogen-activated protein kinases (p-p38) showed a significant increase in LPS-treated immortalized murine microglial cell line BV2 cells in an MPTP-induced mouse model of PD. Knockdown of p62 could suppress the secretion of proinflammatory cytokines and p-p38 of microglia. Besides, inhibition of p38 suppressed the secretion of proinflammatory cytokines and promoted autophagy in BV2 cells. Moreover, our study is the first to identify a unique role of miR-124 in mediating the microglial inflammatory response by targeting p62 and p38 in PD. In the microglial culture supernatant transfer model, the knockdown of p62 in BV2 cells prevented apoptosis and death of human neuroblastoma cell lines SH-SY5Y (SH-SY5Y) cells following microglia activation. In addition, the exogenous delivery of miR-124 could suppress p62 and p-p38 expression and could also attenuate the activation of microglia in the substantia nigra par compacta of MPTP-treated mice. Taken together, our data suggest that miR-124 could inhibit neuroinflammation during the development of PD by targeting p62, p38, and autophagy, indicating that miR-124 could be a potential therapeutic target for regulating the inflammatory response in PD.-Yao, L., Zhu, Z., Wu, J., Zhang, Y., Zhang, H., Sun, X., Qian, C., Wang, B., Xie, L., Zhang, S., Lu, G. MicroRNA-124 regulates the expression of p62/p38 and promotes autophagy in the inflammatory pathogenesis of Parkinson's disease.

Synthesis and biological evaluation of indole-3-carboxamide derivatives as antioxidant agents

Oxidative stress results in various pathologies and as consequence antioxidant agents have attracted uninterrupted attention. In this paper, a novel series of indole-3-carboxamide derivatives (6a–6l) were designed and synthesized based on the melatonin structure as novel antioxidants. All of them were evaluated for the antioxidant activities in vitro against human neuroblastoma SH-SY5Y cell line using H2O2 radical scavenging assay. The target compounds 6a, 6f and 6i indicated better activities than the positive control, ascorbic acid, and 6a exhibited the best antioxidant activity. In addition, the structure-activity relationships of the target compounds were also preliminarily summarized based on the obtained experimental data.