Human Neuroblastoma Cell Line SH-SY5Y Research Articles

Chemical constituents isolated from the aerial parts of Swertia pseudochinensis and their potential neuroprotective effects

Abstract Objective: Swertia pseudochinensis, an annual herb of the genus Swertia in the family Gentianaceae. Some constituents and extracts from the Swertia genus have been recently reported to possess neuroprotective effects, suggesting their potential utility in the prevention and treatment of Parkinson disease (PD). The aim of this work is to identify the chemical constituents and evaluate the potential biological activists of Swertia pseudochinensis. Methods: The phytochemicals from the aerial parts of S. pseudochinensis were isolated and purified by silica gel, Sephadex LH-20 gel, semi-preparative high-performance liquid chromatography, and identified by the spectroscopic methods. All compounds were evaluated for their potential neuroprotective effects against 1-methyl-4-phenylpyridinium-induced apoptosis in the SH-SY5Y human neuroblastoma cell line using the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide assay. Then, we performed an enrichment analysis using the Database for Annotation, Visualization, and Integrated Discovery and investigated the mechanisms by which bellidifolin regulates neurodegenerative disease. Results: Two new anthraquinone, 1,5,6-trimethoxy-2-hydroxy-3-methy-anthraquinone (1) and 1,5,6,8-tetramethoxy-2-hydroxy-3-methyanthraquinone (2), together with nine known including 7-O-β-d-glucopyranosyl-1,8-dihydroxy-3-methoxyxanthone (3), gentisin (4), swertianolin (5), bellidifolin (6), gentiacaulein (7), norswertianolin (8), 5-O-β-d-glucopyranosyl-1,3,8-trihydroxyapatone (9), 1-hydroxy-3,5,8-trimethoxyxanthone (10), and aurantio-obtusin (11), were isolated and compounds 6–8 and 10 exhibited neuroprotective effects at a concentration of 50 μmol/L. Among them, bellidifolin showed significant protective activity, and might have potential as a neuroprotective agent for the treatment of PD, possibly by acting on oxidative damage and reactive oxygen species. Conclusions: These findings indicate that further research on the genus Swertia and its bioactive constituents toward neurodegenerative disorders could be extremely rewarding.

Amphetamine-like stimulants reduce cell viability of human neuroblastoma SH-SY5Y cell line

Amphetamine-like stimulants reduce cell viability of human neuroblastoma SH-SY5Y cell line

HAGLR promotes neuron differentiation through the miR-130a-3p-MeCP2 axis.

Parkinson’s disease (PD) is a prevalent neurodegenerative disease. Currently, the molecular mechanisms underlying the progressions of PD are not fully understood. The human neuroblastoma cell line SH-SY5Y has been widely used as an in vitro model for PD. This study aims to investigate the molecular mechanisms of the non-coding RNA-mediated SH-SY5Y differentiation induced by retinoic acid (RA). By microArray analysis, lncRNA HAGLR was observed to be significantly upregulated during the RA-induced SH-SY5Y differentiation. Silencing HAGLR blocked the RA-induced SH-SY5Y differentiation. Moreover, bioinformatical analysis illustrated that miR-130a-3p contains binding sites for HAGLR. The RNA-pull down assay and luciferase assay demonstrated that HAGLR functioned as a ceRNA of miR-130a-3p in SH-SY5Y cells. Overexpression of miR-130a-3p effectively inhibited SH-SY5Y differentiation. We identified MeCP2, a vital molecule in neuronal diseases, to be a direct target of miR-130a-3p in SH-SY5Y cells by western blot and luciferase assays. The rescue experiments verified that recovery of miR-130a-3p in HAGLR-overexpressing SH-SY5Y cells could successfully overcome the RA-induced SH-SY5Y differentiation by targeting MeCP2. In summary, this study reveals a potential molecular mechanism for the lncRNA-HAGLR-promoted in vitro neuron differentiation by targeting the miR-130a-3p-MeCP2 axis, contributing to the understanding of the pathogenesis and progression of PD.

A Galantamine-Curcumin Hybrid Decreases the Cytotoxicity of Amyloid-Beta Peptide on SH-SY5Y Cells.

Misfolded amyloid beta (Aβ) peptides aggregate and form neurotoxic oligomers. Membrane and mitochondrial damages, calcium dysregulation, oxidative stress, and fibril deposits are among the possible mechanisms of Aβ cytotoxicity. Galantamine (GAL) prevents apoptosis induced by Aβ mainly through the ability to stimulate allosterically the α7 nAChRs and to regulate the calcium cytosolic concentration. Here, we examined the cytoprotective effects of two GAL derivatives, namely compounds 4b and 8, against Aβ cytotoxicity on the human neuroblastoma cell line SH-SY5Y. The protective effects were tested at simultaneous administration, pre-incubation and post-incubation, with Aβ. GAL and curcumin (CU) were used in the study as reference compounds. It was found that 4b protects cells in a similar mode as GAL, while compound 8 and CU potentiate the toxic effects of Aβ. Allosteric stimulation of α7 nAChRs is suggested as a possible mechanism of the cytoprotectivity of 4b. These and previous findings characterize 4b as a prospective non-toxic multi-target agent against neurodegenerative disorders with inhibitory activity on acetylcholinesterase, antioxidant, and cytoprotective properties.

Evaluation and Optimization of Poly-d-Lysine as a Non-Natural Cationic Polypeptide for Gene Transfer in Neuroblastoma Cells

Cationic polypeptides and cationic polymers have cell-penetrating capacities and have been used in gene transfer studies. In this study, we investigate the capability of a polymer of d-lysine (PDL), a chiral form of α–Poly-lysine, as a possible nonviral vector for releasing genetic materials to neuroblastoma cells and evaluate its stability against proteases. We tested and compared its transfection effectiveness in vitro as a vehicle for the EGFP plasmid DNA (pDNA) reporter in the SH-SY5Y human neuroblastoma, HeLa, and 3T3 cell lines. Using fluorescent microscopy and flow cytometry, we demonstrated high transfection efficiencies based on EGFP fluorescence in SH-SY5Y cells, compared with HeLa and 3T3. Our results reveal PDL as an efficient vector for gene delivery specifically in the SH-SY5Y cell line and suggest that PDL can be used as a synthetic cell-penetrating polypeptide for gene therapy in neuroblastoma cells.

RNA-seq analysis reveals significant transcriptome changes in huntingtin-null human neuroblastoma cells

BackgroundHuntingtin (Htt) protein is the product of the gene mutated in Huntington’s disease (HD), a fatal, autosomal dominant, neurodegenerative disorder. Normal Htt is essential for early embryogenesis and the development of the central nervous system. However, the role of Htt in adult tissues is less defined. Following the recent promising clinical trial in which both normal and mutant Htt mRNA were knocked down in HD patients, there is an urgent need to fully understand the molecular consequences of knocking out/down Htt in adult tissues. Htt has been identified as an important transcriptional regulator. Unbiased investigations of transcriptome changes with RNA-sequencing (RNA-Seq) have been done in multiple cell types in HD, further confirming that transcriptional dysregulation is a central pathogenic mechanism in HD. However, there is lack of direct understanding of the transcriptional regulation by normal Htt.MethodsTo investigate the transcriptional role of normal Htt, we first knocked out Htt in the human neuroblastoma SH-SY5Y cell line using the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated protein 9) gene editing approach. We then performed RNA-seq analysis on Htt-null and wild type SH-SY5Y cells to probe the global transcriptome changes induced by Htt deletion.ResultsIn general, Htt has a widespread effect on gene transcription. Functional analysis of the differentially expressed genes (DEGs) using various bioinformatic tools revealed irregularities in pathways related to cell communication and signaling, and more specifically those related to neuron development, neurotransmission and synaptic signaling. We further examined the transcription factors that may regulate these DEGs. Consistent with the disrupted pathways associated with cellular development, we showed that Htt-null cells exhibited slower cell proliferation than wild type cells. We finally validated some of the top DEGS with quantitative RT-PCR.ConclusionsThe widespread transcriptome changes in Htt-null cells could be directly caused by the loss of Htt-mediated transcriptional regulation or due to the secondary consequences of disruption in the gene regulatory network. Our study therefore provides valuable information about key genes associated with Htt-mediated transcription and improves our understanding of the molecular mechanisms underlying the cellular functions of normal and mutant Htt.

GAPDH siRNA Regulates SH-SY5Y Cell Apoptosis Induced by Exogenous α-Synuclein Protein

The transport mechanism of intestinal α-synuclein to the central nervous system has become a new hot topic in Parkinson’s disease (PD) research. It is worth noting that the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been reported to be involved in the pathogenesis of PD. After silencing GAPDH expression by GAPDH siRNA, the normal human intestinal epithelial crypt-like (HIEC) and human SH-SY5Y neuroblastoma cell lines were co-cultured with Escherichia coli cells which were transfected with an α-synuclein overexpression plasmid. The levels of autophagy-related proteins (BECN1, ATG5, LC3A/B and p62) were determined by Western blot analysis. Changes in pro-apoptosis protein levels and flow cytometry analysis were used to assess cell apoptosis and relative intracellular ATP concentration was measured. Oxidative stress was assessed by measuring the levels of reactive oxygen species (ROS) using 2′,7′-dichlorofluorescein diacetate (DCFH-DA), thiobarbituric acid-reactive substances (TBARS), and antioxidant capacity was assessed by measuring the glutathione (GSH) levels and superoxide dismutase (SOD) activity. The silencing of the expression of GAPDH pre-knockdown was found to reduce the intracellular levels of ROS and lipid peroxidation, enhance autophagy activity, thereby reducing the cell injury, apoptosis and necrosis induced by exogenous α-synuclein protein in SH-SY5Y cells. This study identifies a new therapeutic target of exogenous α-synuclein protein induced SH-SY5Y cell injury and improves our understanding of the pathophysiological role of GAPDH in vitro.

Down-Regulation of ID2-AS1 Alleviates the Neuronal Injury Induced by 1-Methy1-4-Phenylpyridinium in Human Neuroblastoma Cell Line SH-SY5Y Cells Through Regulating miR-199a-5p/IFNAR1/JAK2/STAT1 Axis.

We aimed to illustrate the roles and molecular mechanisms of ID2-AS1 in parkinson's disease (PD). Methods: qRT-PCR detected the expression of ID2-AS1. CCK-8, LDH release assays the effect of ID2-AS1 knockdown on PD cells. Flow cytometry and Western Blot were used to detect the effect of ID2-AS1 inhibition on PD cell apoptosis. ELISA analysis showed that ID2-AS1 inhibition can reduce the inflammation of PD cells. ROS activity assay showed that inhibiting ID2-AS1 attenuated the oxidative stress induced by 1-methy1-4-phenylpyridinium (MPP+). RNA binding protein immunoprecipitation assay showed that ID2-AS1 is mainly located in the cytoplasm. The luciferase reporter assay is used to verify the interaction. In our study, ID2-AS1 was concentration-dependently and time-dependently up-regulated in MPP+ -treated human neuroblastoma cell line SH-SY5Y. ID2-AS1 knockdown enhanced cell proliferation and decreased cell death in PD cells. Knockdown of ID2-AS1 attenuates MPP+ -induced cytotoxicity in SH-SY5Y cells. ID2-AS1 is a sponge of miR-199a-5p. IFNAR1 is a target of miR-199a-5p. Inhibition of miR-199a-5p and overexpression of IFNAR1 alleviate the inhibitory effect of ID2-AS1 knockdown on MPP+ triggered neuronal injury. Inhibition of miR-199a-5p and overexpression of IFNAR1 alleviate the inhibitory effect of ID2-AS1 knockdown on MPP+ -triggered JAK2/STAT1 activation. Overall, down-regulation of ID2-AS1 alleviated the neuronal injury in PD through regulating miR-199a-5p/IFNAR1/JAK2/STAT1 axis.

MO140INDOXYL SULFATE INDUCES THE APOPTOSIS OF THE DIFFERENTIATING NEURONS BY ENHANCING OXIDATIVE STRESS AND CLINICAL COGNITIVE IMPAIRMENT

Abstract Background and Aims Indoxyl sulfate (IS) is a protein-bounded uremic toxin involving the multi-organ dysfunction, including neurologic system. It has been noticed that IS could induce the oxidative stress within brain by activating the inflammation mediated by astrocyte, and its concentration was associated with cognitive impairment. It has been noticed that the neuronal progenitor cells with differentiating abilities could replace the damaged neuron. The damage of IS on the progenitor cells with differentiating ability is still lacking. The aim of the study is to investigate the IS mediated cell damage on differentiating neuronal progenitor cells Method The study was divided into 2 parts: clinical investigation and in vitro study. The clinical investigation collected 36 participants (12 health controls and 24 ESRD patients with cognitive impairment by MMSE<=24). The in vitro study used the human neuroblastoma cell line SH-SY5Y cells with differentiating protocol by retinoic acid (RA, with concentration 10%). Indoxyl sulfate was given at concentration at 0, 25, 50 and 100μM at initiation of retinoic acid given and the 7 days after retinoic acid was given. Microscopic morphology, MTT assay, annexin V-PI dual stain, and western blotting for cleaved-caspase 3, Bax, Bcl-2 and oxidative stress (DCFCA) were measured. Results Among the 36 participants, the concentration of IS was higher in the ESRD patients with cognitive impairment (43365.26±24291.55 vs 5212.00±14621.82 mg/dL, p=0.00). The serum concentration of IS was negatively correlated with the total MMSE score (r=-0.446, p=0.006) and the categories of MMSE (orientation to time: r=-0.537, p=0.001, calculation: r=-0.541, p=0.001; recall: r=-0.402,p=0.014). In in vitro study, the cell viability was not influenced in undifferentiated and differentiated (retinoic acid treated for 7 days) SH-SY5Y cells. When IS during the initiation of RA treatment, the cell viability decreased 3 hours after IS was given. The annexin-V-PI dual stain demonstrated that the early apoptotic cells with annexin-V stained increased in IS treated cell with dose-dependent manner. The caspase 8 and cleaved caspase 3 increased at 6 hours after IS treated when RA given. The Bax increased after IS treated at 6 hours. The Bax/Bcl2 ratio increased at the IS concentration 50μM. The DCFCA increased after IS treated at 1 hour with dose-dependent manner, and the cell viability was rescued by N-acetylcystein. Conclusion The serum IS was negatively correlated the MMSE and predictive to cognitive impairment in our cohort with 36 participants (12 health control and 24 ESRD patients with MMSE <=24). The in vitro study demonstrated that IS induced differentiating SH-SY5Y cells apoptosis by activating caspase-dependent cell death after generating oxidative stress.

Isoform-Specific Effects of Apolipoprotein E on Markers of Inflammation and Toxicity in Brain Glia and Neuronal Cells In Vitro.

Mutations to the cholesterol transport protein apolipoprotein E (ApoE) have been identified as a major risk factor for the development of sporadic or late-onset Alzheimer’s disease (AD), with the e4 allele representing an increased risk and the rare e2 allele having a reduced risk compared to the primary e3 form. The reasons behind the change in risk are not entirely understood, though ApoE4 has been connected to inflammation and toxicity in both the brain and the periphery. The goal of this study was to better understand how the ApoE isoforms (ApoE2/3/4) confer differential AD-related risk by assessing cell-specific ApoE-related neuroinflammatory and neurotoxic effects. We compared the effects of ApoE isoforms in vitro on human astrocytes, a human immortalized microglia cell line (HMC3), and the human neuroblastoma cell line SH-SY5Y. Cells were treated for 24 h with or without recombinant ApoE2, ApoE3, or ApoE4 (20 nM) and inflammation and toxicity markers assessed. Our results indicated the expression of inflammatory cytokines IL-1β, TNFα, and IL-6 in human astrocytes was increased in response to all ApoE isoforms, with ApoE4 evoking the highest level of cytokine expression. In response to ApoE2 or ApoE3, microglial cells showed reduced levels of microglial activation markers TREM2 and Clec7a, while ApoE4 induced increased levels of both markers. ApoE2 promoted neuron survival through increased BDNF release from astrocytes. In addition, ApoE2 promoted, while ApoE4 reduced, neuronal viability. Overall, these results suggest that ApoE4 acts on cells in the brain to promote inflammation and neuronal injury and that the deleterious effects of ApoE4 on these cells may, in part, contribute to its role as a risk factor for AD.

SH-SY5Y-derived neurons: a human neuronal model system for investigating TAU sorting and neuronal subtype-specific TAU vulnerability.

The microtubule-associated protein (MAP) TAU is mainly sorted into the axon of healthy brain neurons. Somatodendritic missorting of TAU is a pathological hallmark of many neurodegenerative diseases, including Alzheimer's disease (AD). Cause, consequence and (patho)physiological mechanisms of TAU sorting and missorting are understudied, in part also because of the lack of readily available human neuronal model systems. The human neuroblastoma cell line SH-SY5Y is widely used for studying TAU physiology and TAU-related pathology in AD and related tauopathies. SH-SY5Y cells can be differentiated into neuron-like cells (SH-SY5Y-derived neurons) using various substances. This review evaluates whether SH-SY5Y-derived neurons are a suitable model for (i)investigating intracellular TAU sorting in general, and (ii) with respect to neuron subtype-specific TAU vulnerability. (I) SH-SY5Y-derived neurons show pronounced axodendritic polarity, high levels of axonally localized TAU protein, expression of all six human brain isoforms and TAU phosphorylation similar to the human brain. As SH-SY5Y cells are highly proliferative and readily accessible for genetic engineering, stable transgene integration and leading-edge genome editing are feasible. (II) SH-SY5Y-derived neurons display features of subcortical neurons early affected in many tauopathies. This allows analyzing brain region-specific differences in TAU physiology, also in the context of differential vulnerability to TAU pathology. However, several limitations should be considered when using SH-SY5Y-derived neurons, e.g., thelack of clearly defined neuronal subtypes, or the difficulty of mimicking age-related tauopathy risk factors invitro. In brief, this review discusses the suitability of SH-SY5Y-derived neurons for investigating TAU (mis)sorting mechanisms and neuron-specific TAU vulnerability in disease paradigms.

Effects of antiepileptic drugs’ administration during pregnancy on the nerve cell proliferation and axonal outgrowth of human neuroblastoma SH-SY5Y nerve cells

It has been suggested that the intelligence quotient of children born to pregnant women taking 1000 mg or more of valproic acid per day is lower than that of children born to pregnant women taking other antiepileptic drugs. However, the mechanism whereby intelligence quotient is decreased in children exposed to valproic acid during the fetal period has not yet been elucidated. Therefore, we used the human neuroblastoma cell line SH-SY5Y to evaluate the effects of antiepileptic drugs containing valproic acid on nerve cells. We assessed the anti-proliferative effects of drugs in these cells via WST-8 colorimetric assay, using the Cell Counting Kit-8. We also quantified drug effects on axonal elongation from images using ImageJ software. We also evaluated drug effects on mRNA expression levels on molecules implicated in nervous system development and folic acid uptake using real-time PCR. We observed that carbamazepine and lamotrigen were toxic to SH-SY5Y cells at concentrations >500 μM. In contrast, phenytoin and valproic acid were not toxic to these cells. Carbamazepine, lamotrigen, phenytoin, and valproic acid did not affect axonal outgrowth in SH-SY5Y cells. Sodium channel neuronal type 1a (SCN1A) mRNA expression-level ratios increased when valproic acid was supplemented to cells. The overexpression of SCN1A mRNA due to high valproic acid concentrations during the fetal period may affect neurodevelopment. However, since detailed mechanisms have not yet been elucidated, it is necessary to evaluate it by comparing cell axon elongation and SCN1A protein expression due to high-concentration valproic acid exposure.

ErbB4 regulate extracellular dopamine through the p38 MAPK signaling pathway

ErbB4 loss-of-function in catecholaminergic neurons induces catecholamine dyshomeostasis. Despite ErbB4’s significant role in neuropathology, the signaling pathways that regulate these changes are still widely unknown. In this study, we attempt to identify the downstream pathway of ErbB4 that regulates catecholamine homeostasis. The SH-SY5Y human neuroblastoma cell line was used as the in vitro model for catecholaminergic neurons. Western blotting, enzyme-linked immunosorbent assay, and pharmacological and genetic manipulations by agonist/antagonist or small interference RNA were used to investigate the relationship between ErbB4 and extracellular catecholamines. We confirmed that ErbB4 is abundantly expressed in undifferentiated and retinoic acid-differentiated catecholaminergic cells from the SH-SY5Y cell line. ErbB4 inhibition increase the ratio of phosphorylated p38 to total p38 in SH-SY5Y human neuroblastoma cells. Consistent with previous in vivo observations in mice, ErbB4 deficiency led to increases in extracellular dopamine and norepinephrine levels. However, the resulting increase in extracellular dopamine, but not norepinephrine, could be suppressed by p38 inhibitor SB202190. Our results suggest that both extracellular dopamine and norepinephrine homeostasis could be regulated by ErbB4 in human catecholaminergic cells, and ErbB4 may regulate extracellular dopamine, but not norepinephrine, through the p38 MAPK signaling pathway, thus indicating different regulatory pathways of dopamine and norepinephrine by ErbB4 in catecholaminergic neurons.

Stable DNMT3L overexpression in SH-SY5Y neurons recreates a facet of the genome-wide Down syndrome DNA methylation signature

BackgroundDown syndrome (DS) is characterized by a genome-wide profile of differential DNA methylation that is skewed towards hypermethylation in most tissues, including brain, and includes pan-tissue differential methylation. The molecular mechanisms involve the overexpression of genes related to DNA methylation on chromosome 21. Here, we stably overexpressed the chromosome 21 gene DNA methyltransferase 3L (DNMT3L) in the human SH-SY5Y neuroblastoma cell line and assayed DNA methylation at over 26 million CpGs by whole genome bisulfite sequencing (WGBS) at three different developmental phases (undifferentiated, differentiating, and differentiated).ResultsDNMT3L overexpression resulted in global CpG and CpG island hypermethylation as well as thousands of differentially methylated regions (DMRs). The DNMT3L DMRs were skewed towards hypermethylation and mapped to genes involved in neurodevelopment, cellular signaling, and gene regulation. Consensus DNMT3L DMRs showed that cell lines clustered by genotype and then differentiation phase, demonstrating sets of common genes affected across neuronal differentiation. The hypermethylated DNMT3L DMRs from all pairwise comparisons were enriched for regions of bivalent chromatin marked by H3K4me3 as well as differentially methylated sites from previous DS studies of diverse tissues. In contrast, the hypomethylated DNMT3L DMRs from all pairwise comparisons displayed a tissue-specific profile enriched for regions of heterochromatin marked by H3K9me3 during embryonic development.ConclusionsTaken together, these results support a mechanism whereby regions of bivalent chromatin that lose H3K4me3 during neuronal differentiation are targeted by excess DNMT3L and become hypermethylated. Overall, these findings demonstrate that DNMT3L overexpression during neurodevelopment recreates a facet of the genome-wide DS DNA methylation signature by targeting known genes and gene clusters that display pan-tissue differential methylation in DS.

Palmitic acid promotes resistin-induced insulin resistance and inflammation in SH-SY5Y human neuroblastoma

Saturated fatty acids such as palmitic acid promote inflammation and insulin resistance in peripheral tissues, contrasting with the protective action of polyunsaturated fatty acids such docosahexaenoic acid. Palmitic acid effects have been in part attributed to its potential action through Toll-like receptor 4. Beside, resistin, an adipokine, also promotes inflammation and insulin resistance via TLR4. In the brain, palmitic acid and resistin trigger neuroinflammation and insulin resistance, but their link at the neuronal level is unknown. Using human SH-SY5Yneuroblastoma cell line we show that palmitic acid treatment impaired insulin-dependent Akt and Erk phosphorylation whereas DHA preserved insulin action. Palmitic acid up-regulated TLR4 as well as pro-inflammatory cytokines IL6 and TNFα contrasting with DHA effect. Similarly to palmitic acid, resistin treatment induced the up-regulation of IL6 and TNFα as well as NFκB activation. Importantly, palmitic acid potentiated the resistin-dependent NFkB activation whereas DHA abolished it. The recruitment of TLR4 to membrane lipid rafts was increased by palmitic acid treatment; this is concomitant with the augmentation of resistin-induced TLR4/MYD88/TIRAP complex formation mandatory for TLR4 signaling. In conclusion, palmitic acid increased TLR4 expression promoting resistin signaling through TLR4 up-regulation and its recruitment to membrane lipid rafts.

Cannabidiol induces autophagy via ERK1/2 activation in neural cells

Autophagy is a lysosomal catabolic process essential to cell homeostasis and is related to the neuroprotection of the central nervous system. Cannabidiol (CBD) is a non-psychotropic phytocannabinoid present in Cannabis sativa. Many therapeutic actions have been linked to this compound, including autophagy activation. However, the precise underlying molecular mechanisms remain unclear, and the downstream functional significance of these actions has yet to be determined. Here, we investigated CBD-evoked effects on autophagy in human neuroblastoma SH-SY5Y and murine astrocyte cell lines. We found that CBD-induced autophagy was substantially reduced in the presence of CB1, CB2 and TRPV1 receptor antagonists, AM 251, AM 630 and capsazepine, respectively. This result strongly indicates that the activation of these receptors mediates the autophagic flux. Additionally, we demonstrated that CBD activates autophagy through ERK1/2 activation and AKT suppression. Interestingly, CBD-mediated autophagy activation is dependent on the autophagy initiator ULK1, but mTORC1 independent. Thus, it is plausible that a non-canonical pathway is involved. Our findings collectively provide evidence that CBD stimulates autophagy signal transduction via crosstalk between the ERK1/2 and AKT kinases, which represent putative regulators of cell proliferation and survival. Furthermore, our study sheds light on potential therapeutic cannabinoid targets that could be developed for treating neurodegenerative disorders.

Biochemical characterization of proliferative and differentiated SH-SY5Y cell line as a model for Parkinson's disease

Parkinson's disease is a multifactorial neurodegenerative disease. The cellular pathology includes dopamine depletion, decrease in mitochondrial complex I enzyme activity, lysosomal glucocerebrosidase enzyme activity and glutathione levels. The SH-SY5Y human neuroblastoma cell line is one of the most widely used cell line models for Parkinson's disease. However, the consensus on its suitability as a model in its proliferative or differentiated state is lacking. In this study, we characterized and compared the biochemical processes most often studied in PD. This in proliferative and differentiated phenotypes of SH-SY5Y cells and several differences were found. Most notably, extracellular dopamine metabolism was significantly higher in differentiated SH-SY5Y. Furthermore, there was a greater variability in glutathione levels in proliferative phenotype (+/- 49%) compared to differentiated (+/- 16%). Finally, enzyme activity assay revealed significant increase in the lysosomal enzyme glucocerebrosidase activity in differentiated phenotype. In contrast, our study has found similarities between the two phenotypes in mitochondrial electron transport chain activity and tyrosine hydroxylase protein expression. The results of this study demonstrate that despite coming from the same cell line, these cells possess some key differences in their biochemistry. This highlights the importance of careful characterization of relevant disease pathways to assess the suitability of cell lines, such as SH-SY5Y cells, for modelling PD or other diseases, i.e. when using the same cell line but different differentiation states.

The Effects of Serum Removal on Gene Expression and Morphological Plasticity Markers in Differentiated SH-SY5Y Cells

Despite the widespread use of the SH-SY5Y human neuroblastoma cell line in modeling human neurons in vitro, protocols for growth, differentiation and experimentation differ considerably across the literature. Many studies fully differentiate SH-SY5Y cells before experimentation, to investigate plasticity measures in a mature, human neuronal-like cell model. Prior to experimentation, serum is often removed from cell culture media, to arrest the cell growth cycle and synchronize cells. However, the exact effect of this serum removal before experimentation on mature, differentiated SH-SY5Y cells has not yet been described. In studies using differentiated SH-SY5Y cells, any effect of serum removal on plasticity markers may influence results. The aim of the current study was to systematically characterize, in differentiated, neuronal-like SH-SY5Y cells, the potentially confounding effects of complete serum removal in terms of morphological and gene expression markers of plasticity. We measured changes in commonly used morphological markers and in genes related to neuroplasticity and synaptogenesis, particularly in the BDNF-TrkB signaling pathway. We found that complete serum removal from already differentiated SH-SY5Y cells increases neurite length, neurite branching, and the proportion of cells with a primary neurite, as well as proportion of βIII-Tubulin and MAP2 expressing cells. Gene expression results also indicate increased expression of PSD95 and NTRK2 expression 24 h after serum removal. We conclude that serum deprivation in differentiated SH-SY5Y cells affects morphology and gene expression and can potentially confound plasticity-related outcome measures, having significant implications for experimental design in studies using differentiated SH-SY5Y cells as a model of human neurons.

New malonylginsenosides from the fresh fruits of Panax notoginseng

Four new malonylginsenosides, malonylnotoginsenoside Fe (1), malonylnotoginsenoside Ra1 (2), malonylgypenoside LXXV (3), and malonylginsenoside Mc (4), together with two known analogues, malonylfloralginsenoside Rc1 (5) and malonylginsenoside Rc (6), were isolated from the fresh fruits of Panax notoginseng. Their structures were determined by MS and NMR experiments. The anti-proliferative activities of the malonylginsenosides (1–6) against SH-SY5Y human neuroblastoma cell line were evaluated using the MTT assay.

Phosphorylation of Specificity Protein 3 Is Critical for Activation of β4-Galactosyltransferase 3 Gene Promoter in SH-SY5Y Human Neuroblastoma Cell Line.

Elevated expression of β4-galactosyltransferase (β4GalT) 3 is correlated with poor clinical outcome of neuroblastoma patients. Our recent study has revealed that the transcription of the β4GalT3 gene is activated by Specificity protein (Sp) 3 in SH-SY5Y human neuroblastoma cell line. Here we report the biological significance of the Sp3 phosphorylation in the transcriptional activation of the β4GalT3 gene. The treatment of SH-SY5Y cells with 10% fetal bovine serum (FBS) increased the mitogen-activated protein kinase (MAPK) signaling and the promoter activity of the β4GalT3 gene. Meanwhile, the treatment with U0126, an inhibitor for MAPK kinase, decreased the MAPK signaling and the promoter activity. These findings indicate that the transcriptional activation of the β4GalT3 gene is mediated by the MAPK signaling. In SH-SY5Y cells cultured in the medium containing 10% FBS, the serine (Ser) residues in Sp3 were phosphorylated. Human Sp3 contains four Ser residues, Ser73, Ser563, Ser566, and Ser646, as the putative phosphorylation sites. Sp3 mutant with the mutation of Ser73 did not decrease the promoter activation of the β4GalT3 gene, indicating that Ser73 is uninvolved in the promoter activation of the β4GalT3 gene by Sp3. In contrast, Sp3 mutants with the mutations of Ser563, Ser566, and Ser646 significantly reduced the promoter activation by Sp3. The results suggest that the phosphorylation of these Ser residues is implicated in the promoter activation by Sp3. This study demonstrates that the phosphorylation of Sp3 plays important roles in the transcriptional activation of the β4GalT3 gene in human neuroblastoma.