Human platelet lysate supports SH-SY5Y neuroblastoma cell proliferation and differentiation into a dopaminergic-like neuronal phenotype under xenogeneic-free culture conditions.

SH-SY5Y is a human neuroblastoma cell line that can be differentiated into several neuronal phenotypes, depending on culture conditions. For this reason, this cell line has been widely used as an in vitro model of neurodegenerative conditions, such as Parkinson's disease (PD). However, most studies published to date used fetal bovine serum (FBS) as culture medium supplement for SH-SY5Y cell differentiation. We report on the testing of human platelet lysate (hPL) as a culture medium supplement to support SH-SY5Y cell culture. Both standard hPL and a fibrinogen-depleted hPL (FD-hPL) formulation, which does not require the addition of anticoagulants to culture media, promoted an increase in SH-SY5Y cell proliferation in comparison to FBS, without compromising metabolic activity. SH-SY5Y cells cultured in hPL or FD-hPL also displayed a higher number of neurite extensions and stained positive for MAP2 and synaptophysin, in the absence of differentiation stimuli; reducing hPL or FD-hPL concentration to 1% v/v did not affect cell proliferation or metabolic activity. Furthermore, following treatment with retinoic acid (RA) and further stimulation with brain-derived neurotrophic factor (BDNF) and nerve growth factor beta (NGF-ß), the percentage of SH-SY5Y cells stained positive for dopaminergic neuronal differentiation markers (tyrosine hydroxylase [TH] and Dopamine Transporter [DAT]) was higher in hPL or FD-hPL than in FBS, and gene expression of dopaminergic markers TH, DAT, and DR2 was also detected. Overall, the data herein presented supports the use of hPL to differentiate SH-SY5Y cells into a neuronal phenotype with dopaminergic features, and the adoption of FD-hPL as a fully xenogeneic free alternative to FBS to support the use of SH-SY5Y cells as a neurodegeneration model.

Lysophosphatidic Acid Stimulates Mitogenic Activity and Signaling in Human Neuroblastoma Cells through a Crosstalk with Anaplastic Lymphoma Kinase.

Lysophosphatidic acid (LPA) is a well-documented pro-oncogenic factor in different cancers, but relatively little is known on its biological activity in neuroblastoma. The LPA effects and the participation of the tyrosine kinase receptor anaplastic lymphoma kinase (ALK) in LPA mitogenic signaling were studied in human neuroblastoma cell lines. We used light microscopy and [3H]-thymidine incorporation to determine cell proliferation, Western blot to study intracellular signaling, and pharmacological and molecular tools to examine the role of ALK. We found that LPA stimulated the growth of human neuroblastoma cells, as indicated by the enhanced cell number, clonogenic activity, and DNA synthesis. These effects were curtailed by the selective ALK inhibitors NPV-TAE684 and alectinib. In a panel of human neuroblastoma cell lines harboring different ALK genomic status, the ALK inhibitors suppressed LPA-induced phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2), which are major regulators of cell proliferation. ALK depletion by siRNA treatment attenuated LPA-induced ERK1/2 activation. LPA enhanced ALK phosphorylation and potentiated ALK activation by the ALK ligand FAM150B. LPA enhanced the inhibitory phosphorylation of the tumor suppressor FoxO3a, and this response was impaired by the ALK inhibitors. These results indicate that LPA stimulates mitogenesis of human neuroblastoma cells through a crosstalk with ALK.

Lipid levels correlate with neuronal and dopaminergic markers during the differentiation of SH-SY5Y cells.

Parkinson's Disease (PD) is characterised by the loss of dopaminergic neurons and the deposition of protein inclusions called Lewy Bodies (LBs). LBs are heterogeneous structures composed of protein and lipid molecules and their main constituent is the presynaptic protein α-synuclein. SH-SY5Y cells are neuroblastoma cells commonly used to model PD because they express dopaminergic markers and α-synuclein and they can be differentiated into neuronal cells using established protocols. Despite increasing evidence pointing towards a role of lipids in PD, limited knowledge is available on the lipidome of undifferentiated and differentiated SH-SY5Y cells. Using a combination of lipidomics, proteomics, morphological and electrophysiological measurements, we identified specific lipids, including sphingolipids, whose levels are affected by the differentiation of SH-SY5Y neuroblastoma cells and found that the levels of these lipids correlate with those of neuronal and dopaminergic markers. These results provide a quantitative characterisation of the changes in lipidome associated with the differentiation of SH-SY5Y cells into more neuronal and dopaminergic-like phenotype and serve as a basis for further characterisation of lipid disruptions in association with PD and its risk factors in this dopaminergic-like neuronal cell model.

Ruthenium(II) Polypyridyl Complexes with Benzoxazole Derivatives and Non-Innocent Ligands as Effective Antioxidants in Human Neuroblasts.

Ruthenium(II) polypyridyl complexes continue to raise increasing interest for the encouraging results in several biomedical areas. Considering their vast chemical-physical repertoire, in particular the possibility to switch from the sensitization of reactive oxygen species (ROS) to ROS-scavenging abilities by tuning the nature of their ligands, it is therefore surprising that their potential as antioxidants has not been largely investigated so far. Herein, we explored the antioxidant behaviour of the novel ruthenium compound [Ru(dbpy)(2,3-DAN)Cl]PF6 (Ru1), featuring a benzoxazole derivative (dpby=2,6-bis(4-methyl-2-benzoxazolyl)pyridine) and the non-innocent 2,3-diamminonaftalene (2,3-DAN) ligand, along with the reference tpy-containing analogue [Ru(tpy)(2,3-DAN)Cl]PF6 (Ru2) (tpy=2,2':6',2''-terpyridine). Following the synthesis and the electrochemical characterization, chemical antioxidant assays highlighted the beneficial role of dpby for the ROS-scavenging properties of Ru1. These data have been corroborated by the highest protective effect of Ru1 against the oxidative stress induced in SH-SY5Y human neuroblastoma, which exerts pro-survival and anti-inflammatory actions. The results herein reported highlight the potential of Ru1 as pharmacological tool in neurodegenerative diseases and specially prove that the antioxidant properties of such compounds are likely the result of a non-trivial synergetic action involving the bioactive ligands in their chemical architectures.

Development of a Three-Dimensional Multicellular Model of Human Neuroblastoma Using Matrigel as an Extracellular Matrix Analogue.

Neuroblastoma, the most prevalent extracranial solid tumor in children, poses therapeutic challenges due to its variable clinical course and propensity for metastasis. Despite advances in treatment strategies like chemotherapy, drug resistance remains a significant concern, highlighting the need for improved models to study tumor behavior and drug responses. This chapter proposes the development of a three-dimensional multicellular model of human neuroblastoma using Matrigel as an ECM analogue. Such models aim to replicate the complexity of the tumor microenvironment, providing valuable insights into tumor progression and drug resistance mechanisms. By recapitulating key features of neuroblastoma within a physiologically relevant context, these models offer a platform for preclinical drug screening and therapeutic development.

Cholesterol Modulation Attenuates the AD-like Phenotype Induced by Herpes Simplex Virus Type 1 Infection.

Cholesterol, a crucial component of cell membranes, influences various biological processes, including membrane trafficking, signal transduction, and host-pathogen interactions. Disruptions in cholesterol homeostasis have been linked to congenital and acquired conditions, including neurodegenerative disorders such as Alzheimer's disease (AD). Previous research from our group has demonstrated that herpes simplex virus type I (HSV-1) induces an AD-like phenotype in several cell models of infection. This study explores the interplay between cholesterol and HSV-1-induced neurodegeneration. The impact of cholesterol was determined by modulating its levels with methyl-beta-cyclodextrin (MßCD) using the neuroblastoma cell lines SK-N-MC and N2a. We have found that HSV-1 infection triggers the intracellular accumulation of cholesterol in structures resembling endolysosomal/autophagic compartments, a process reversible upon MßCD treatment. Moreover, MßCD exhibits inhibitory effects at various stages of HSV-1 infection, underscoring the importance of cellular cholesterol levels, not only in the viral entry process but also in subsequent post-entry stages. MßCD also alleviated several features of AD-like neurodegeneration induced by viral infection, including lysosomal impairment and intracellular accumulation of amyloid-beta peptide (Aß) and phosphorylated tau. In conclusion, these findings highlight the connection between cholesterol, neurodegeneration, and HSV-1 infection, providing valuable insights into the underlying mechanisms of AD.

Novel combination of Olesoxime/Resveratrol-encapsulated exosomes to improve cognitive function by targeting amyloid ß-induced Alzheimer's disease: investigation on in vitro and in vivo model.

Alzheimer's disease (AD) is a fatal neurological illness that worsens with time. Preventing the aggregate formation of amyloid beta protein is a promising approach to treat Alzheimer's disease. This article describes an amiable procedure for the synthesis of Olesoxime-Resveratrol (OLX-RSV) encapsulated in exosomes. By suppressing Aß1-42 aggregation and crossing the blood-brain barrier also known as BBB after intravenous treatment without resulting in any discernible damage, the nanocomposite demonstrated good biocompatibility. A variety of characterization technique including particle size, TEM, and in vitro drug release experiments, were used to characterize the exosomes. Human Neuroblastoma (SHSY5Y) cells were used to test the cytotoxicity and viability of cells of the formulation using the Cell Counting Kit-8 assay. The prepared OLX-RSV-loaded exosomes were tested for their ability to suppress Aß1-42 in SHSY5Y Cells by analyzing the amyloid samples using CD spectra. The effects of apoptosis on Human neuroblastoma cells were studied using cytofluorometry. The parameters of SOD, caspase-3 and the ability to scavenge reactive oxygen species (ROS) were also evaluated. The behavioral outcomes of Morris water maze test demonstrated that OLX-RSV-loaded exosomes significantly enhanced the APP/PS1 mice's capacity to learn and remember spatial cues. Therefore, we hypothesized that OLX-RSV-loaded exosomes could be a useful and efficient method in the treatment of AD.

Mistranslating the genetic code with leucine in yeast and mammalian cells.

Translation fidelity relies on accurate aminoacylation of transfer RNAs (tRNAs) by aminoacyl-tRNA synthetases (AARSs). AARSs specific for alanine (Ala), leucine (Leu), serine, and pyrrolysine do not recognize the anticodon bases. Single nucleotide anticodon variants in their cognate tRNAs can lead to mistranslation. Human genomes include both rare and more common mistranslating tRNA variants. We investigated three rare human tRNALeu variants that mis-incorporate Leu at phenylalanine or tryptophan codons. Expression of each tRNALeu anticodon variant in neuroblastoma cells caused defects in fluorescent protein production without significantly increased cytotoxicity under normal conditions or in the context of proteasome inhibition. Using tRNA sequencing and mass spectrometry we confirmed that each tRNALeu variant was expressed and generated mistranslation with Leu. To probe the flexibility of the entire genetic code towards Leu mis-incorporation, we created 64 yeast strains to express all possible tRNALeu anticodon variants in a doxycycline-inducible system. While some variants showed mild or no growth defects, many anticodon variants, enriched with G/C at positions 35 and 36, including those replacing Leu for proline, arginine, alanine, or glycine, caused dramatic reductions in growth. Differential phenotypic defects were observed for tRNALeu mutants with synonymous anticodons and for different tRNALeu isoacceptors with the same anticodon. A comparison to tRNAAla anticodon variants demonstrates that Ala mis-incorporation is more tolerable than Leu at nearly every codon. The data show that the nature of the amino acid substitution, the tRNA gene, and the anticodon are each important factors that influence the ability of cells to tolerate mistranslating tRNAs.

Oleoylethanolamide and Palmitoylethanolamide Enhance IFNß-Induced Apoptosis in Human Neuroblastoma SH-SY5Y Cells.

Oleoylethanolamide (OEA) and palmitoylethanolamide (PEA) are endogenous lipids that act as agonists of the peroxisome proliferator-activated receptor α (PPARα). Recently, an interest in the role of these lipids in malignant tumors has emerged. Nevertheless, the effects of OEA and PEA on human neuroblastoma cells are still not documented. Type I interferons (IFNs) are immunomodulatory cytokines endowed with antiviral and anti-proliferative actions and are used in the treatment of various pathologies such as different cancer forms (i.e., non-Hodgkin's lymphoma, melanoma, leukemia), hepatitis B, hepatitis C, multiple sclerosis, and many others. In this study, we investigated the effect of OEA and PEA on human neuroblastoma SH-SY5Y cells treated with IFNß. We focused on evaluating cell viability, cell proliferation, and cell signaling. Co-exposure to either OEA or PEA along with IFNß leads to increased apoptotic cell death marked by the cleavage of caspase 3 and poly-(ADP ribose) polymerase (PARP) alongside a decrease in survivin and IKBα levels. Moreover, we found that OEA and PEA did not affect IFNß signaling through the JAK-STAT pathway and the STAT1-inducible protein kinase R (PKR). OEA and PEA also increased the phosphorylation of p38 MAP kinase and programmed death-ligand 1 (PD-L1) expression both in full cell lysate and surface membranes. Furthermore, GW6471, a PPARα inhibitor, and the genetic silencing of the receptor were shown to lower PD-L1 and cleaved PARP levels. These results reveal the presence of a novel mechanism, independent of the IFNß-prompted pathway, by which OEA and PEA can directly impair cell survival, proliferation, and clonogenicity through modulating and potentiating the intrinsic apoptotic pathway in human SH-SY5Y cells.

Assessment of Protective Effects of DTPA, NAC, and Taurine on Possible Cytotoxicity Induced by Individual and Combined Zinc Oxide and Copper Oxide Nanoparticles in SH-SY5Y Cells.

The present study investigated the cytotoxic effects of ZnO, CuO, and mixed combinations of them on SH-SY5Y cells. For this purpose, the cells were exposed to various concentrations of these NPs alone for 24-96 h and as a mixture for 24 h. Variations in cell viability were noted. MTT results showed that ZnO and/or CuO NPs decreased cell survival by about 59% at 200 (ZnO, at 24 h) and 800 µg/ml (ZnO and/or CuO, at 72 and 96 h). When the NR assay was used, slight decreases were noted with ZnO NPs at 72 and 96 h. With CuO NPs alone and NPs in a mixture, only the highest concentrations caused 40 and 70% decreases in cell survival, respectively. Especially with NR assays, DTPA, NAC, or taurine provided marked protection. ROS levels were increased with the highest concentration of CuO NPs and with all concentrations of the mixture. The highest concentration of ZnO NPs and the lowest concentration of CuO NPs caused slight decreases in mitochondrial membrane potential levels. Additionally, increases were noted in caspase 3/7 levels with ZnO and CuO NPs alone or with a mixture of them. Intracellular calcium levels were decreased in this system. These findings demonstrated that ZnO and CuO NPs, either separately or in combination, had a modest cytotoxic effect on SH-SY5Y cells. Protection obtained with DTPA, NAC, or taurine against the cytotoxicity of these NPs and the ROS-inducing effect of CuO NPs and the NPs' mixture suggests that oxidative stress might be involved in the cytotoxicity mechanisms of these NPs.

Molecular docking analysis of chlorpyrifos at the human α7-nAChR and its potential relationship with neurocytoxicity in SH-SY5Y cells.

The organophosphate insecticide chlorpyrifos (CPF), an acetylcholinesterase inhibitor, has raised serious concerns about human safety. Apart from inducing synaptic acetylcholine accumulation, CPF could also act at nicotinic acetylcholine receptors, like the α7-isoform (α7-nAChR), which could potentially be harmful to developing brains. Our aims were to use molecular docking to assess the binding interactions between CPF and α7-nAChR through, to test the neurocytotoxic and oxidative effects of very low concentrations of CPF on SH-SY5Y cells, and to hypothesize about the potential mediation of α7-nAChR. Docking analysis showed a significant binding affinity of CPH for the E fragment of the α7-nAChR (ΔGibbs: -5.63 to -6.85 Kcal/mol). According to the MTT- and Trypan Blue-based viability assays, commercial CPF showed concentration- and time-dependent neurotoxic effects at a concentration range (2.5-20 µM), ten-folds lower than those reported to have crucial effects for sheer CPF. A rise of the production of radical oxygen species (ROS) was seen at even lower concentrations (1-2.5 µM) of CPF after 24h. Notably, our docking analysis supports the antagonistic actions of CPF on α7-nAChR that were recently published. In conclusion, while α7-nAChR is responsible for neuronal survival and neurodevelopmental processes, its activity may also mediate the neurotoxicity of CPF.

Silencing of the PHLDA1 leads to global proteome changes and differentiation pathways of human neuroblastoma cells.

Neuroblastoma (NB) is the most common extracranial pediatric solid tumor originating from the abnormal development of cells of the sympathoadrenal lineage of the neural crest. Targeting GD2 ganglioside (GD2), a glycolipid expressed on neuroblastoma cells, with GD2 ganglioside-recognizing antibodies affects several pivotal signaling routes that drive or influence the malignant phenotype of the cells. Previously performed gene expression profiling helped us to identify the PHLDA1 (pleckstrin homology-like domain family A member 1) gene as the most upregulated gene in the IMR-32 human neuroblastoma cells treated with the mouse 14G2a monoclonal antibody. Mass spectrometry-based proteomic analyses were applied to better characterize a role of PHLDA1 protein in the response of neuroblastoma cells to chimeric ch14.18/CHO antibody. Additionally, global protein expression profile analysis in the IMR-32 cell line with PHLDA1 silencing revealed the increase in biological functions of mitochondria, accompanied by differentiation-like phenotype of the cells. Moreover, mass spectrometry analysis of the proteins co-immunoprecipitated using anti-PHLDA1-specific antibody, selected a group of possible PHLDA1 binding partners. Also, a more detailed analysis suggested that PHLDA1 interacts with the DCAF7/AUTS2 complex, a key component of neuronal differentiation in vitro. Importantly, our results indicate that PHLDA1 silencing enhances the EGF receptor signaling pathway and combinatory treatment of gefitinib and ch14.18/CHO antibodies might be beneficial for neuroblastoma patients. Data are available via ProteomeXchange with the identifier PXD044319.

2,3,7,8-Tetrachlorodibenzo-p-dioxin and kynurenine induce Parkin expression in neuroblastoma cells through different signaling pathways mediated by the aryl hydrocarbon receptor.

Parkin regulates protein degradation and mitophagy in dopaminergic neurons. Deficiencies in Parkin expression or function lead to cellular stress, cell degeneration, and the death of dopaminergic neurons, which promotes Parkinson's disease. In contrast, Parkin overexpression promotes neuronal survival. Therefore, the mechanisms of Parkin upregulation are crucial to understand. We describe here the molecular mechanism of AHR-mediated Parkin regulation in human SH-SY5Y neuroblastoma cells. Specifically, we report that the human Parkin gene (PRKN) is transcriptionally upregulated by the aryl hydrocarbon receptor (AHR) through two different selective ligand-dependent pathways. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a stress-inducing AHR ligand, indirectly promotes PRKN transcription by inducing ATF4 expression via TCDD-mediated endoplasmic reticulum (ER) stress. In contrast, kynurenine, a nontoxic AHR agonist, induces PRKN transcription by promoting AHR binding to the PRKN promoter without activating ER stress. Our results demonstrate that AHR activation may be a potential pharmacological pathway to induce human Parkin, but such a strategy must carefully consider the choice of AHR ligand to avoid neurotoxic side effects.

Cell adhesion molecule immobilized gold surfaces for enhanced neuron-electrode interfaces.

To provide a long-term solution for increasing the biocompatibility of neuroprosthetics, approaches to reduce the side effects of invasive neuro-implantable devices are still in need of improvement. Physical, chemical, and bioactive design aspects of the biomaterials are proven to be important for providing proper cell-to-cell, cell-to-material interactions. Particularly, modification of implant surfaces with bioactive cues, especially cell adhesion molecules (CAMs) that capitalize on native neural adhesion mechanisms, are promising candidates in favor of providing efficient interfaces. Within this concept, this study utilized specific CAMs, namely N-Cadherin (Neural cadherin, N-Cad) and neural cell adhesion molecule (NCAM), to enhance neuron-electrode contact by mimicking the cell-to-ECM interactions for improving the survival of cells and promoting neurite outgrowth. For this purpose, representative gold electrode surfaces were modified with N-Cadherin, NCAM, and the mixture (1:1) of these molecules. Modifications were characterized, and the effect of surface modification on both differentiated and undifferentiated neuroblastoma SH-SY5Y cell lines were compared. The findings demonstrated the successful modification of these molecules which subsequently exhibited biocompatible properties as evidenced by the cell viability results. In cell culture experiments, the CAMs displayed promising results in promoting neurite outgrowth compared to conventional poly-l-lysine coated surfaces, especially NCAM and N-Cad/NCAM modified surfaces clearly showed significant improvement. Overall, this optimized approach is expected to provide an insight into the action mechanisms of cells against the local environment and advance processes for the fabrication of alternative neural interfaces.

Ethyl pyruvate attenuates cerebral hypoxia/reoxygenation injury in neuroblastoma cells: Role of GAS6/Axl signaling.

Background: Ischemic stroke, caused neurological dysfunction due to inadequate blood supply to brain, has a high morbidity and mortality. Ethyl pyruvate (EP), a simple aliphatic ester derived from pyruvic acid, has the advantages of safety and stability. Studies have confirmed that EP has anti-oxidative, anti-inflammation, anti-tumor, and other pharmacological effects, and it demonstrates significant therapeutic effects on multiple diseases. GAS6 and its high affinity Axl receptor play an important role in cell adhesion, anti-apoptosis, proliferation and migration by activating downstream signal transduction pathways. Previous studies have demonstrated the neuroprotective effects of the GAS6/Axl axis. Methods: A series of experimental methods were employed to confirm the effect of EP against cerebral hypoxia/reoxygenation (HR) injury. Results: In this study, the protective effect and mechanism of EP on HR injury in N2a cells was explored. The results found that treatment with EP could increase HR-injured neuronal viability, improve cell morphology, and reduce LDH release and ROS accumulation, thereby exhibiting a neuroprotective effect. Furthermore, EP treatment restored the down-regulated expression of GAS6, Axl, NQO1, PGC-1α, NRF1, and UCP2 caused by HR injury. Specifically, it was observed that the neuroprotective effect of EP was partially inhibited by GAS6 siRNA. Conclusion: In conclusion, these results suggest that EP treatment attenuates HR-induced oxidative stress injury in neuroblastoma cells via activating GAS6/Axl signaling.

Nordihydroguaiaretic Acid Affects Undifferentiated and Differentiated Neuroblastoma Cells Differently through Mechanisms that Impact on Cell Viability.

AIM: We aimed to investigate the mechanisms involved in the neurotoxic effects of NDGA on differentiated and undifferentiated human neuroblastoma cells (MSN), assessing cell viability, changes in the actin cytoskeleton, cell migration and the expression of the 5-LOX enzyme and the inhibitor of cell cycle progression p21WAF1/CIP1. BACKGROUND: High expression and activity of the lipoxygenase enzyme (LOX) have been detected in several tumors, including neuroblastoma samples, suggesting the use of LOX inhibitors as potential therapy molecules. Among these, the natural compound nordihydroguaiaretic acid (NDGA) has been extensively tested as an antiproliferative drug against diverse types of cancer cells. OBJECTIVE: In this study, we analyzed the toxic effect of NDGA on neuroblastoma cells at a dose that did not affect cell survival when they differentiated to a neuron-like phenotype and the potential mechanisms involved in the anticancer properties. METHODS: We exposed human neuroblastoma cells (MSN) to different concentrations of NDGA before and after a differentiation protocol with retinoic acid and nerve growth factor and analyzed cell viability, cell migration, actin cytoskeleton morphology and the levels of the cell cycle inhibitor p21WAF1/CIP1 and 5-LOX. RESULTS: We found that differentiated human neuroblastoma cells are more resistant to NDGA than undifferentiated cells. The toxic effects of NDGA were accompanied by reduced cell migration, changes in actin cytoskeleton morphology, induction of p21WAF1/CIP1 and decreased levels of the 5- LOX enzyme. CONCLUSION: This study provides new evidence regarding the potential use of NDGA to induce cell death in human neuroblastoma.

Initiation of Brain Extract Fibrillation and Effective Cellular Internalization of Tryptophan Fibrils Unveils Its Neurotoxicity Risk.

Recent discoveries on the self-assembly of aromatic amino acids into amyloid-like neurotoxic nanostructures have initiated a quest to decode the molecular mechanisms for the initiation of neurodegeneration. Moreover, the multicomponent nature of the amyloid deposits still questions the existing and well-defined amyloid cascade hypothesis. Hence, deciphering the neurotoxicity of amyloid-like nanostructures of aromatic amino acids becomes crucial for understanding the etiology of amyloidogenesis. Here, we demonstrate the cellular internalization and consequential damaging effects of self-assembled amyloid-like tryptophan nanostructures on human neuroblastoma cells. The cell-damaging potential of tryptophan nanostructure seems to be facilitated via ROS generation, necrosis and apoptosis mediated cell death. Further, tryptophan nanostructures were found to be seeding competent conformers, which triggered aggressive aggregation of brain extract components. The early stage intermediate nanostructures possess a higher cross-seeding efficacy than the seeding potential of the matured tryptophan fibrils. In addition to the cell-damaging and cross-seeding effects, tryptophan fibrils were found to catalyze oxidation of neuromodulator dopamine. These findings add more insights into the specific role of tryptophan self-assembly during the pathogenesis of hypertryptophanemia and other amyloid-associated neurodegenerative complications.

Deletion of the Homocysteine Thiolactone Detoxifying Enzyme Bleomycin Hydrolase, in Mice, Causes Memory and Neurological Deficits and Worsens Alzheimer's Disease-Related Behavioral and Biochemical Traits in the 5xFAD Model of Alzheimer's Disease.

BACKGROUND: Bleomycin hydrolase (BLMH), a homocysteine (Hcy)-thiolactone detoxifying enzyme, is attenuated in Alzheimer's disease (AD) brains. Blmh loss causes astrogliosis in mice while the loss of histone demethylase Phf8, which controls mTOR signaling, causes neuropathy in mice and humans. OBJECTIVE: To examine how Blmh gene deletion affects the Phf8/H4K20me1/mTOR/autophagy pathway, amyloid-ß (Aß) accumulation, and cognitive/neuromotor performance in mice. METHODS: We generated a new mouse model of AD, the Blmh-/-5xFAD mouse. Behavioral assessments were conducted by cognitive/neuromotor testing. Blmh and Phf8 genes were silenced in mouse neuroblastoma N2a-APPswe cells by RNA interference. mTOR- and autophagy-related proteins, and AßPP were quantified by western blotting and the corresponding mRNAs by RT-qPCR. Aß was quantified by western blotting (brains) and by confocal microscopy (cells). RESULTS: Behavioral testing showed cognitive/neuromotor deficits in Blmh-/- and Blmh-/-5xFAD mice. Phf8 was transcriptionally downregulated in Blmh-/- and Blmh-/-5xFAD brains. H4K20me1, mTOR, phospho-mTOR, and AßPP were upregulated while autophagy markers Becn1, Atg5, and Atg7 were downregulated in Blmh-/- and Blmh-/-5xFAD brains. Aß was elevated in Blmh-/-5xFAD brains. These biochemical changes were recapitulated in Blmh-silenced N2a-APPswe cells, which also showed increased H4K20me1-mTOR promoter binding and impaired autophagy flux (Lc3-I, Lc3-II, p62). Phf8-silencing or treatments with Hcy-thiolactone or N-Hcy-protein, metabolites elevated in Blmh-/- mice, induced biochemical changes in N2a-APPswe cells like those induced by the Blmh-silencing. However, Phf8-silencing elevated Aß without affecting AßPP. CONCLUSIONS: Our findings show that Blmh interacts with AßPP and the Phf8/H4K20me1/mTOR/autophagy pathway, and that disruption of those interactions causes Aß accumulation and cognitive/neuromotor deficits.

Functional Interaction between Adenosine A2A and mGlu5 Receptors Mediates STEP Phosphatase Activation and Promotes STEP/mGlu5R Binding in Mouse Hippocampus and Neuroblastoma Cell Line.

(1) Background: Recently, we found that adenosine A2A receptor (A2AR) stimulation results in an increase in STEP phosphatase activity. In order to delve into the mechanism through which A2AR stimulation induced STEP activation, we investigated the involvement of mGlu5R since it is well documented that A2AR and mGlu5R physically and functionally interact in several brain areas. (2) Methods: In a neuroblastoma cell line (SH-SY5Y) and in mouse hippocampal slices, we evaluated the enzymatic activity of STEP by using a para-nitrophenyl phosphate colorimetric assay. A co-immunoprecipitation assay and a Western blot analysis were used to evaluate STEP/mGlu5R binding. (3) Results: We found that the A2AR-dependent activation of STEP was mediated by the mGlu5R. Indeed, the A2AR agonist CGS 21680 significantly increased STEP activity, and this effect was prevented not only by the A2AR antagonist ZM 241385, as expected, but also by the mGlu5R antagonist MPEP. In addition, we found that mGlu5R agonist DHPG-induced STEP activation was reversed not only by the mGlu5R antagonist MPEP but also by ZM 241385. Finally, via co-immunoprecipitation experiments, we found that mGlu5R and STEP physically interact when both receptors are activated (4) Conclusions: These results demonstrated a close functional interaction between mGlu5 and A2A receptors in the modulation of STEP activity.

Protective and Therapeutic Role of Ginkgo Biloba Extract Through TRPV1 Channels in an in Vitro Alzheimer's Disease Model.

Introduction: The effect of Ginkgo biloba (GB) on mitochondria-dependent TRPV1 ion channels in neuroblastoma cells was investigated by creating an Alzheimer's disease (AD) model. Methods: Okadaic acid was applied on SH-SY5Y cells to create an AD model. After cellular differentiation, the study was organized with the seven main groups, examining the effect of GB on calcium depended TRPV1 channels in neuroblastoma cells AD, has been established in vitro. Results: The higher Ca2+ concentration was detected in the GB+AD, AD and AD+GB groups when compared with the control (p<0.001). The Ca2+ level was lower in GB+AD and AD+GB groups than in the AD group (p<0.001). Also, cytosolic Ca2+ concentration was lower in the GB+AD than in the AD+GB group (p<0.05), the apoptosis and intracellular reactive oxygen species (ROS) values were higher in the GB+AD, AD and AD+GB groups than in the control (p<0.001). The apoptosis and intracellular ROS values were higher in AD group than in the GB+AD and AD+GB group (p<0.001) and the apoptosis level was higher in AD+GB group than GB+AD group (p<0.001) and the mitochondrial depolarization, caspase 3 and caspase 9 levels were higher in the GB+AD, AD and AD+GB groups when compared to the control group (p<0.001). Also, the values were lower in the GB+AD group, AD group and AD+GB groups when compared with the GB+AD+capsazepine group, AD+capsazepine group and AD+GB+capsazepine respectively (p<0.001). Conclusion: These results show us that GB has a protective effect besides its therapeutic effect in Alzheimer's disease via TRPV1 channel.