Glutamic Acid Modified Gold Nanorod Sensor for the Detection of Calcium ions in Neuronal Cells.

Calcium (Ca2+) ions play a crucial role in the functioning of neurons, governing various aspects of neuronal activity such as rapid modulation and alterations in gene expression. Ca2+ signaling has a significant impact on the development of diseases and the impairment of neuronal functions. Herein, the study reports a Ca2+ ion sensor in neuronal cells using a gold nanorod. The gold nanorod (GA-GNR) conjugated glutamic acid developed in the study was used as a nano-bio probe for the experimental and in vitro detection of calcium. The nanosensor is colloidally stable, preserves plasmonic properties, and shows good viability in neuronal cells, as well as promoting neuron cell line growth. The cytotoxicity and cell penetration of the nanosensor are studied using Raman spectroscopy, brightfield and darkfield microscopy imaging, and MTT assays. The quantification of Ca2+ ions in neuronal cells is determined by monitoring the surface plasmon resonance (SPR) of the GA-GNR. The change in the intensity profile in the presence of Ca2+ incubated neurons was effectively used to develop a portable prototype of an optical Ca2+ sensor, proposing it as a tool for neurodegenerative disease diagnosis and neuromodulation evaluation.

Creation of an in vitro model of GM1 gangliosidosis by CRISPR/Cas9 knocking-out the GLB1 gene in SH-SY5Y human neuronal cell line.

GM1 gangliosidosis is one type of hereditary error of metabolism that occurs due to the absence or reduction of ß-galactosidase enzyme content in the lysosome of cells, including neurons. In vitro, the use of neural cell lines could facilitate the study of this disease. By creating a cell model of GM1 gangliosidosis on the SH-SY5Y human nerve cell line, it is possible to understand the main role of this enzyme in breaking down lipid substrate and other pathophysiologic phenomena this disease. To knock-out the human GLB1 gene, guides targeting exons 14 and 16 of the GLB1 gene were designed using the CRISPOR and CHOP-CHOP websites, and high-efficiency guides were selected for cloning in the PX458 vector. After confirming the cloning, the vectors were transformed into DH5α bacteria and then the target vector was extracted and transfected into human nerve cells (SH-SY5Y cell line) by electroporation. After 48 h, GFP+ cells were sorted using the FACS technique and homozygous (compound heterozygous) single cells were isolated using the serial dilution method and sequencing was done to confirm them. Finally, gap PCR tests, X-gal and Periodic acid-Schiff (PAS) staining, and qPCR were used to confirm the knock-out of the human GLB1 gene. Additionally, RNA sequencing data analysis from existing data of the Gene Expression Omnibus (GEO) was used to find the correlation of GLB1 with other genes, and then the top correlated genes were tested for further evaluation of knock-out effects. The nonviral introduction of two guides targeting exons 14 and 16 of the GLB1 gene into SH-SY5Y cells led to the deletion of a large fragment with a size of 4.62 kb. In contrast to the non-transfected cell, X-gal staining resulted in no blue color in GLB1 gene knock-out cells indicating the absence of ß-galactosidase enzyme activity in these cells. Real-time PCR (qPCR) results confirmed the RNA-Seq analysis outcomes on the GEO data set and following the GLB1 gene knock-out, the expression of its downstream genes, NEU1 and CTSA, has been decreased. It has been also shown that the downregulation of GLB1-NEU1-CTSA complex gene was involved in suppressed proliferation and invasion ability of knock-out cells. This study proved that using dual guide RNA can be used as a simple and efficient tool for targeting the GLB1 gene in nerve cells and the knockout SH-SY5Y cells can be used as a model investigation of basic and therapeutic surveys for GM1 gangliosidosis disease.

Spinetoram-Induced Potential Neurotoxicity through Autophagy Mediated by Mitochondrial Damage.

Spinetoram is an important semi-synthetic insecticide extensively applied in agriculture. It is neurotoxic to insects, primarily by acting on acetylcholine receptors (nAChRs). However, few studies have examined the neurotoxicity of spinetoram in human beings. In this study, various concentrations (5, 10, 15, and 20 µM) of spinetoram were employed to expose SH-SY5Y cells in order to study the neurotoxic effects of spinetoram. The results showed that spinetoram exposure markedly inhibited cell viability and induced oxidative stress. It also induced mitochondrial membrane potential collapse (ΔΨm), and then caused a massive opening of the mitochondrial permeability transition pore (mPTP), a decrease in ATP synthesis, and Ca2+ overloading. Furthermore, spinetoram exposure induced cellular autophagy, as evidenced by the formation of autophagosomes, the conversion of LC3-I into LC3-II, down-regulation of p62, and up-regulation of beclin-1. In addition, we observed that p-mTOR expression decreased, while p-AMPK expression increased when exposed to spinetoram, indicating spinetoram triggered AMPK/mTOR-mediated autophagy. Complementarily, the effect of spinetoram on neurobehavior was studied using the zebrafish model. After being exposed to different concentrations (5, 10, and 20 µg/mL) of spinetoram, zebrafish showed neurobehavioral irregularities, such as reduced frequency of tail swings and spontaneous movements. Similarly, autophagy was also observed in zebrafish. In conclusion, spinetoram exposure produced potential neurotoxicity through autophagy mediated by mitochondrial damage. The experimental data and results of the neurotoxicity study of spinetoram provided above are intended to serve as reference for its safety assessment.

SH-SY5Y Cell Line In Vitro Models for Parkinson Disease Research-Old Practice for New Trends.

The SH-SY5Y cell line is a simple and inexpensive in vitro experimental model for studying Parkinson disease (PD). This experimental model is a useful tool for elucidating pathophysiological mechanisms of PD and in the development of new pharmacological therapies. In this review, we aim to summarize current protocols for SH-SY5Y cell culturing and differentiation and PD experimental designs derived from the SH-SY5Y cell line. The most efficient protocol for differentiation of the SH-SY5Y cell line into dopaminergic neurons seems to be the addition of retinoic acid to the growth medium, followed by 12-O-tetradecanoylphorbol-13-acetate (TPA) addition in a low concentration of fetal bovine serum. PD pathological changes, such as neuronal apoptosis and the intraneuronal alpha-synuclein aggregation, can be reproduced in the SH-SY5Y cell line either by the use of neurotoxic agents [such as rotenone, 1-methyl-4-phenylpyridinium (MPP+), 6-hydroxydopamine] or by genetic modification (transfection of the alpha-synuclein wild-type or mutant gene, genetic manipulation of other genes involved in PD). In addition, compounds with a potential neuroprotective role may be tested on neurotoxicity-induced SH-SY5Y models. The cell line can also be used for testing PD pathophysiological mechanisms such as the prion-like neuronal transmission of alpha-synuclein or the microbiota influence in PD. In conclusion, the use of the SH-SY5Y cell line represents a basic but consistent first step in experiments related to PD, but which must be followed by the confirmation of the results through more complex in vitro and in vivo experimental models.

A Comparative Study between Lycorine and Galantamine Abilities to Interact with AMYLOID ß and Reduce In Vitro Neurotoxicity.

Galantamine is a natural alkaloid extracted from the Amaryllidaceae plants and is used as the active ingredient of a drug approved for the treatment of the early stages of Alzheimer's disease. It mainly acts as an acetylcholinesterase (AChE) inhibitor, increasing concentrations of the acetylcholine neurotransmitter. Recent cellular studies have also shown the ability of galantamine to protect SH-SY5Y cell lines against amyloid-ß (Aß)-induced toxicity. Such investigations have supported and validated further in-depth studies for understanding the chemical and molecular features associated with galantamine-protective abilities. In addition to galantamine, other natural alkaloids are known to possess AChE inhibitory activity; among them lycorine has been extensively investigated for its antibacterial, anti-inflammatory and antitumoral activities as well. Despite its interesting biological properties, lycorine's neuroprotective functions against Aß-induced damages have not been explored so far. In this research study, the ability of galantamine and lycorine to suppress Aß-induced in vitro neuronal toxicity was evaluated by investigating the chemical interactions of the two alkaloids with Aß peptide. A multi-technique spectroscopic analysis and cellular cytotoxicity assays were applied to obtain new insights on these molecular associations. The comparison between the behaviors exhibited by the two alkaloids indicates that both compounds possess analogue abilities to interact with the amyloidogenic peptide and protect cells.

Cell Cycle Reactivation, at the Start of Neurodegeneration, Induced by Forskolin and Aniline in Differentiated Neuroblastoma Cells.

A characteristic hallmark of Alzheimer's disease (AD) is the intracellular accumulation of hyperphosphorylated tau protein, a phenomenon that appears to have associations with oxidative stress, double-stranded DNA breakage, and the de-condensation of heterochromatin. Re-entry into the cell division cycle appears to be involved in the onset of this neurodegenerative process. Indeed, the cell cycle cannot proceed regularly in the differentiated neurons leading to cell death. Here, we induced cell cycle reactivation in neuronal-like cells, obtained by neuroblastoma cells treated with retinoic acid, by exposure to forskolin or aniline. These compounds determine tau hyperphosphorylation or oxidative stress, respectively, resulting in the appearance of features resembling the start of neuronal degeneration typical of AD, such as tau hyperphosphorylation and re-entry into the cell cycle. Indeed, we detected an increased transcriptional level of cyclins and the appearance of a high number of mitotic cells. We also observed a delay in the initiation of the cell cycle when forskolin was co-administered with pituitary adenylate cyclase-activating polypeptide (PACAP). This delay was not observed when PACAP was co-administered with aniline. Our data demonstrate the relevance of tau hyperphosphorylation in initiating an ectopic cell cycle in differentiated neuronal cells, a condition that can lead to neurodegeneration. Moreover, we highlight the utility of neuroblastoma cell lines as an in vitro cellular model to test the possible neuroprotective effects of natural molecules.

Indoles and 1-(3-(benzyloxy)benzyl)piperazines: Reversible and selective monoamine oxidase B inhibitors identified by screening an in-house compound library.

The therapeutic indications for monoamine oxidases A and B (MAO-A and MAO-B) inhibitors that have emerged from biological studies on animal and cellular models of neurological and oncological diseases have focused drug discovery projects upon identifying reversible MAO inhibitors. Screening of our in-house academic compound library identified two hit compounds that inhibit MAO-B with IC50 values in micromolar range. Two series of indole (23 analogues) and 3-(benzyloxy)benzyl)piperazine (16 analogues) MAO-B inhibitors were derived from hits, and screened for their structure-activity relationships. Both series yielded low micromolar selective inhibitors of human MAO-B, namely indole 2 (IC50 = 12.63 ± 1.21 µM) and piperazine 39 (IC50 = 19.25 ± 4.89 µM), which is comparable to selective MAO-B inhibitor isatin (IC50 = 6.10 ± 2.81 µM), yet less potent in comparison to safinamide (IC50 = 0.029 ± 0.002 µM). Selective MAO-B inhibitors 2, 14, 38 and 39 exhibited favourable permeation of the blood-brain barrier and low cytotoxicity in the human neuroblastoma cell line SH-SY5Y.

The Impact of Differentiation on Cytotoxicity and Insulin Sensitivity in Streptozotocin Treated SH-SY5Y Cells.

Recently neuronal insulin resistance was suggested playing a role in Alzheimer's disease. Streptozotocin (STZ) is commonly used to induce impairment in insulin metabolism. In our previous work on undifferentiated SH-SY5Y cells the compound exerted cytotoxicity without altering insulin sensitivity. Nevertheless, differentiation of the cells to a more mature neuron-like phenotype may considerably affect the significance of insulin signaling and its sensitivity to STZ. We aimed at studying the influence of STZ treatment on insulin signaling in SH-SY5Y cells differentiated by retinoic acid (RA). Cytotoxicity of STZ or low serum (LS) condition and protective effect of insulin were compared in RA differentiated SH-SY5Y cells. The effect of insulin and an incretin analogue, exendin-4 on insulin signaling was also examined by assessing glycogen synthase kinase-3 (GSK-3) phosphorylation. STZ was found less cytotoxic in the differentiated cells compared to our previous results in undifferentiated SH-SY5Y cells. The cytoprotective concentration of insulin was similar in the STZ and LS groups. However, the right-shifted concentration-response curve of insulin induced GSK-3 phosphorylation in STZ-treated differentiated cells is suggestive of the development of insulin resistance that was further confirmed by the insulin potentiating effect of exendin-4. Differentiation reduced the sensitivity of SH-SY5Y cells for the non-specific cytotoxicity of STZ and enhanced the relative significance of development of insulin resistance. The differentiated cells thus serve as a better model for studying the role of insulin signaling in neuronal survival. However, direct cytotoxicity of STZ also contributes to the cell death.

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

Misfolded amyloid beta (Aß) peptides aggregate and form neurotoxic oligomers. Membrane and mitochondrial damages, calcium dysregulation, oxidative stress, and fibril deposits are among the possible mechanisms of Aß cytotoxicity. Galantamine (GAL) prevents apoptosis induced by Aß 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 Aß cytotoxicity on the human neuroblastoma cell line SH-SY5Y. The protective effects were tested at simultaneous administration, pre-incubation and post-incubation, with Aß. 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 Aß. 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.

Lack of insulin resistance in response to streptozotocin treatment in neuronal SH-SY5Y cell line.

Recently, it is suggested that brain insulin resistance may contribute to the development of Alzheimer's disease; therefore, there is a high interest in its investigation. Streptozotocin (STZ) is often used to induce dysregulation of glucose and insulin metabolism in animal and cell culture models. Alteration in insulin sensitivity however, has not yet been assessed in neuronal cells after STZ treatment. We aimed at studying the concentration dependence of the protective effect of insulin on STZ-induced damage using SH-SY5Y cell line. Cells were treated with STZ and cell viability was assessed by resazurin reduction and lactate dehydrogenase release assays. Low serum (LS) medium was used as control damage. The effect of various concentrations (30, 100, 300, 1000 nM) of insulin was studied on cell viability and glycogen synthase kinase-3 (GSK-3) phosphorylation, an indicator of insulin signaling. STZ induced dose- and time-dependent cytotoxicity, its 1 mM concentration exerted a low, gradually developing damage. The cytoprotective effect of insulin was demonstrated in both STZ and LS groups. Its maximal effect was lower in the STZ-treated cells; however, its effective concentration remained largely unaltered. Insulin-induced GSK-3 phosphorylation was similar in the STZ- and LS-treated cells suggesting unchanged insulin signaling. Our present results indicate that STZ does not induce significant impairment in insulin sensitivity in SH-SY5Y cells, thus in this cell line it is not a good tool for studying the role of insulin resistance in neurodegeneration and to examine protective agents acting by improving insulin signaling.

Netrin-1 protects the SH-SY5Y cells against amyloid beta neurotoxicity through NF-κB/Nrf2 dependent mechanism.

Many evidence confirms that amyloid beta 1-42 fragment (Aß1-42) causes neuroinflammation, oxidative stress, and cell death, which are related to progressive memory loss, cognitive impairments and mental disorders that will lead to Alzheimer's disease (AD) progression. Netrin-1, as a member of the laminins, has been proved to inhibit apoptosis and inflammation outside of nervous system, in addition to having a vital role in morphogenesis and neurogenesis of neural system. This study was designed to assess the protective effects of netrin-1 in SH-SY5Y human neuroblastoma cell line exposed to Aß1-42 and to explore some mechanisms that underlie netrin-1 effects. Cultured SH-SY5Y neuroblast-like cells were treated with netrin-1 prior to Aß1-42 exposure and the effects were assessed by MTT and ELISA assay kits. Netrin- 1 pretreatment of Aß1-42-exposed SH-SY5Y human neuroblastoma cells attenuated Aß1-42 induced toxic effects, increased cell viability and partially restored levels of 3 inflammatory and oxidative stress biomarkers including: nuclear factor erythroid 2-like 2 (Nrf2), tumor necrosis factor alpha (TNFα) and nuclear factor kappa-light chain-enhancer of activated B cells (NF-κB). Based on the findings of this study, netrin-1 represents a promising therapeutic bio agent to abrogate cellular inflammation and reactive oxygen species (ROS) activation induced by Aß1-42 in the SH-SY5Y cell model of AD.

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 24 h. However, after a longer exposure time (72 h), 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.

Growth-promoting effects of low-level butyl benzyl phthalate exposure on human neuroblastoma SH-SY5Y cells.

The purpose of present study was to investigate the impact of butyl benzyl phthalate (BBP) on SH-SY5Y neuroblastoma cells in vitro. The cell counting kit-8 was used to measure cell proliferation and flow cytometry was utilized to study cell cycle phases and apoptosis. Western blotting and quantitative real-time polymerase chain reaction were used to detect levels of aromatase, estrogen receptors (ERs) and some apoptosis and cell cycle-related genes. Results showed BBP-stimulated SH-SY5Y cells in a dose-dependent manner and produced a reverted U-shaped dose-response curve. BBP at lower concentrations (0.01 and 0.1 µm) significantly induced cell proliferation while inhibited cell growth at 300 µm. The promoting effect of estradiol could be entirely blocked by administration of ICI182 780, a pure antagonist of ERs, while the effect of BBP could be partly blocked. Additionally, we confirmed 0.1 µm BBP-induced cell proliferation caused the arrest of cells in S phase and inhibited apoptosis, which might be partially explained by the decreased expression of p53, the increased expression of proliferating cell nuclear antigen, Bcl-2 and cell cycle regulator cyclin-D1, and the activation of aromatase. The addition of ICI182 780 had no effect on BBP-induced ERß mRNA expression, whereas ICI182 780 could effectively counteract the effect of estradiol. Moreover, pretreatment with ICI182 780 could block the induction of aromatase protein expression and activity by BBP, showing an involvement of ERs. Except for the ER pathway, these results showed there might be other pathways involved in promoting the effects of low-level BBP on SH-SY5Y cells, which require further investigation.

Homocysteine induces PUMA-mediated mitochondrial apoptosis in SH-SY5Y cells.

Previous studies have reported that homocysteine induced endoplasmic reticulum (ER) stress in neuronal cells, proposing the underlying mechanism by which it could induce neurotoxicity. Induction of pro-apoptotic transcription factor C/EBP homologous protein (CHOP) and activation of caspase-4 by calpain have been suggested to be an important route in inducing apoptosis in response to ER stress. In this study, we investigated the molecular pathway of homocysteine-induced apoptosis in caspase-4 deficient SH-SY5Y human neuroblastoma cells. Homocysteine significantly increased mRNA levels of CHOP and p53, resulting in the upregulation of their downstream target gene, p53 up-regulated modulator of apoptosis (PUMA). In cells treated with homocysteine, Bcl-2-associated X protein (BAX) protein levels, cytochrome c release from the mitochondria, and caspase-9 activation were significantly increased. Consistently, a caspase-9 inhibitor significantly alleviated homocysteine-induced cytotoxicity. Significantly lower BAX mRNA levels and caspase-9 activation were observed in cells transfected with siRNA for PUMA. Taken together, our findings suggest that PUMA would be involved in the possible crosstalk between the ER and the mitochondria in the homocysteine-induced apoptosis of caspase-4 deficient SH-SY5Y cells.

Impact of diamond nanoparticles on neural cells.

Diamond nanoparticles (DNPs) are very attractive for biomedical applications, particularly for bioimaging. The aim of this study was to evaluate the impact of DNPs on neural cancer cells and thus to assess the possible application of DNPs for these cells imaging. For this purpose, the neuroblastoma SH-SY5Y cell line was chosen. Cells were cultured in medium with different concentrations (15, 50, 100 and 150 µg/ml) of DNPs. After 48 h of incubation, cell metabolic activity was evaluated by the XTT assay. For assessment of cellular metabolic activity, cells were also cultured on differently terminated nanocrystalline diamond (NCD) coatings in medium with 150 µg/ml of DNPs. Cell adhesion and morphology were evaluated by brightfield microscopy. Diamond nanoparticle internalization was determined by confocal microscopy. The obtained results showed that low concentrations (15, 50 and 100 µg/ml) of nanoparticles did not significantly affect the SH-SY5Y cell metabolic activity. However, a higher concentration (150 µg/ml) of DNPs statistically significantly reduced SH-SY5Y cell metabolic activity. After 48 h incubation with 150 µg/ml DNPs, cell metabolic activity was 23% lower than in medium without DNPs on standard tissue culture polystyrene.

Chitosan-Tricarbocyanine-Based Nanogels Were Able to Cross the Blood-Brain Barrier Showing Its Potential as a Targeted Site Delivery Agent.

Targeting drugs to the central nervous system (CNS) is challenging due to the presence of the blood-brain barrier (BBB). The cutting edge in nanotechnology generates optimism to overcome the growing challenges in biomedical sciences through the effective engineering of nanogels. The primary objective of the present report was to develop and characterize a biocompatible natural chitosan (CS)-based NG that can be tracked thanks to the tricarbocyanine (CNN) fluorescent probe addition on the biopolymer backbone. FTIR shed light on the chemical groups involved in the CS and CNN interactions and between CNN-CS and tripolyphosphate, the cross-linking agent. Both in vitro and in vivo experiments were carried out to determine if CS-NGs can be utilized as therapeutic delivery vehicles directed towards the brain. An ionic gelation method was chosen to generate cationic CNN-CS-NG. DLS and TEM confirmed that these entities' sizes fell into the nanoscale. CNN-CS-NG was found to be non-cytotoxic, as determined in the SH-SY5Y neuroblastoma cell line through biocompatibility assays. After cellular internalization, the occurrence of an endo-lysosomal escape (a crucial event for an efficient drug delivery) of CNN-CS-NG was detected. Furthermore, CNN-CS-NG administered intraperitoneally to female CF-1 mice were detected in different brain regions after 2 h of administration, using fluorescence microscopy. To conclude, the obtained findings in the present report can be useful in the field of neuro-nanomedicine when designing drug vehicles with the purpose of delivering drugs to the CNS.

Study of cytotoxicity in neuroblastoma cell line exposed to patulin and citrinin.

Mycotoxins can be found in food and feed storage as well as in several kinds of foodstuff and are capable of harming mammals and some of them even in small doses. This study investigated on the undifferentiated neuronal cell line SH-SY5Y the effects of two mycotoxins: patulin (PAT) and citrinin (CTN), which are predominantly produced by fungi species Penicillium and Aspergillus. Here, the individual and combined cytotoxicity of PAT and CTN was investigated using the cytotoxic assay MTT. Our findings indicate that after 24 h of treatment, the IC50 value for PAT is 2.01 µM, which decreases at 1.5 µM after 48 h. In contrast, CTN did not attain an IC50 value at the tested concentration. Therefore, we found PAT to be the more toxic compared to CTN. However, the combined treatment suggests an additive toxic effect. With 2,7-dichlorodihydrofluorescin diacetate (DCFH-DA) DCFH-DA assay, ROS generation was demonstrated after CTN treatment, but PAT showed only small changes. The mixture presented a very constant behavior over time. Finally, the median-effect/combination index (CI-) isobologram equation demonstrated an additive effect after 24 h, but an antagonistic effect after 48 h for the interaction of the two mycotoxins.

The SH-SY5Y cell line: a valuable tool for Parkinson's disease drug discovery.

INTRODUCTION: Owing to limited efficient treatment strategies for highly prevalent and distressing Parkinson's disease (PD), an impending need emerged for deciphering new modes and mechanisms for effective management. SH-SY5Y-based in vitro neuronal models have emerged as a new possibility for the elucidation of cellular and molecular processes in the pathogenesis of PD. SH-SY5Y cells are of human origin, adhered to catecholaminergic neuronal attributes, which consequences in imparting wide acceptance and significance to this model over conventional in vitro PD models for high-throughput screening of therapeutics. AREAS COVERED: Herein, the authors review the SH-SY5Y cell line and its value to PD research. The authors also provide the reader with their expert perspectives on how these developments can lead to the development of new impactful therapeutics. EXPERT OPINION: Encouraged by recent research on SH-SY5Y cell lines, it was envisaged that this in vitro model can serve as a primary model for assessing efficacy and toxicity of new therapeutics as well as for nanocarriers' capacity in delivering therapeutic agents across BBB. Considering the proximity with human neuronal environment as in pathogenic PD conditions, SH-SY5Y cell lines vindicated consistency and reproducibility in experimental results. Accordingly, exploitation of this standardized SH-SY5Y cell line can fast-track the drug discovery and development path for novel therapeutics.

Exploring the Interaction of Indole-3-Acetonitrile with Neuroblastoma Cells: Understanding the Connection with the Serotonin and Dopamine Pathways.

Indole-3-acetonitrile, a compound produced by bacteria and plants as a defense and survival signal in response to attacks, has been recently discovered as a metabolite produced by human cancer cells. This discovery suggests a potential association between IAN and cancer progression in patients. Consequently, the aim of this work was to study the effects of IAN on a specific cancer cell line, SH-SY5Y, and elucidate its connection to the serotonin and dopamine pathways by examining the precursors of these neurotransmitters. To achieve this, a cellular viability assay was conducted, along with a morphological evaluation of the cells under both normal and stress conditions. Our results demonstrated that for the highest concentrations in our study, IAN was able to reduce the cellular viability of the cells. Furthermore, when IAN was combined with the amino acids that originate the neurotransmitters, it was possible to observe that in both combinations there was a decrease in the viability of the cells. Thus, IAN may in fact have some influence on both the serotonin and dopamine pathways since changes in cell viability were observed when it was added together with the amino acids. This preliminary study indicates the presence of an interaction between IAN and neuroblastoma cells that justifies further exploration and study.

In Vitro Approaches for the Study of Pneumococcal-Neuronal Interaction and Pathogenesis.

CFU- and confocal microscopy-based in vitro methods to assess pneumococcal adhesion and invasion of relevant human cells, such as neurons, remain a powerful tool to understand the basis of host-pathogen interactions. In recent years, there has been a continuous refinement of confocal detection of human and bacterial cells through the use of specific, fluorochrome-labelled antibodies. Used in combination, these assays provide both the means for quantification and enough flexibility to accommodate specific experimental needs.