A new advanced cellular model of functional cholinergic-like neurons developed by reprogramming the human SH-SY5Y neuroblastoma cell line.

Modeling human neuronal properties in physiological and pathological conditions is essential to identify novel potential drugs and to explore pathological mechanisms of neurological diseases. For this purpose, we generated a three-dimensional (3D) neuronal culture, by employing the readily available human neuroblastoma SH-SY5Y cell line, and a new differentiation protocol. The entire differentiation process occurred in a matrix and lasted 47 days, with 7 days of pre-differentiation phase and 40 days of differentiation, and allowed the development of a 3D culture in conditions consistent with the physiological environment. Neurons in the culture were electrically active, were able to establish functional networks, and showed features of cholinergic neurons. Hence here we provide an easily accessible, reproducible, and suitable culture method that might empower studies on synaptic function, vesicle trafficking, and metabolism, which sustain neuronal activity and cerebral circuits. Moreover, this novel differentiation protocol could represent a promising cellular tool to study physiological cellular processes, such as migration, differentiation, maturation, and to develop novel therapeutic approaches.

Cannabidiol Antiproliferative Effect in Triple-Negative Breast Cancer MDA-MB-231 Cells Is Modulated by Its Physical State and by IGF-1.

Cannabidiol (CBD) is a non-psychoactive phytocannabinoid that has been discussed for its safety and efficacy in cancer treatments. For this reason, we have inquired into its use on triple-negative human breast cancer. Analyzing the biological effects of CBD on MDA-MB-231, we have demonstrated that both CBD dosage and serum concentrations in the culture medium influence its outcomes; furthermore, light scattering studies demonstrated that serum impacts the CBD aggregation state by acting as a surfactant agent. Pharmacological studies on CBD in combination with chemotherapeutic agents reveal that CBD possesses a protective action against the cytotoxic effect exerted by cisplatin on MDA-MB-231 grown in standard conditions. Furthermore, in a low serum condition (0.5%), starting from a threshold concentration (5 µM), CBD forms aggregates, exerts cytostatic antiproliferative outcomes, and promotes cell cycle arrest activating autophagy. At doses above the threshold, CBD exerts a highly cytotoxic effect inducing bubbling cell death. Finally, IGF-1 and EGF antagonize the antiproliferative effect of CBD protecting cells from harmful consequences of CBD aggregates. In conclusion, CBD effect is strongly associated with the physical state and concentration that reaches the treated cells, parameters not taken into account in most of the research papers.

Green and Roasted Coffee Extracts Inhibit Interferon-ß Release in LPS-Stimulated Human Macrophages.

The anti-inflammatory activity of coffee extracts is widely recognized and supported by experimental evidence, in both in vitro and in vivo settings, mainly murine models. Here, we investigated the immunomodulatory properties of coffee extracts from green (GCE) and medium-roasted (RCE) Coffea canephora beans in human macrophages. The biological effect of GCE and RCE was characterized in LPS-stimulated THP-1-derived human macrophages (TDM) as a model of inflammation. Results showed decreased amounts of TNF-α, IL-6 and IL-1ß and a strong dose-dependent inhibition of interferon-ß (IFN-ß) release. Molecular mechanism of IFN-ß inhibition was further investigated by immunofluorescence confocal microscopy analysis that showed a diminished nuclear translocation of p-IRF-3, the main transcription factor responsible for IFN-ß synthesis. The inhibition of IFN-ß release by RCE and GCE was also confirmed in human primary CD14+ monocytes-derived macrophages (MDM). The main component of coffee extracts, 5-O-caffeoylquinic acid (5-CQA) also inhibited IFN-ß production, through a mechanism occurring downstream to TLR4. Inhibition of IFN-ß release by coffee extracts parallels with the activity of their main phytochemical component, 5-CQA, thus suggesting that this compound is the main responsible for the immunomodulatory effect observed. The application of 5-CQA and coffee derived-phytoextracts to target interferonopathies and inflammation-related diseases could open new pharmacological and nutritional perspectives.

RalGPS2 Interacts with Akt and PDK1 Promoting Tunneling Nanotubes Formation in Bladder Cancer and Kidney Cells Microenvironment.

RalGPS2 is a Ras-independent Guanine Nucleotide Exchange Factor for RalA GTPase that is involved in several cellular processes, including cytoskeletal organization. Previously, we demonstrated that RalGPS2 also plays a role in the formation of tunneling nanotubes (TNTs) in bladder cancer 5637 cells. In particular, TNTs are a novel mechanism of cell-cell communication in the tumor microenvironment, playing a central role in cancer progression and metastasis formation. However, the molecular mechanisms involved in TNTs formation still need to be fully elucidated. Here we demonstrate that mid and high-stage bladder cancer cell lines have functional TNTs, which can transfer mitochondria. Moreover, using confocal fluorescence time-lapse microscopy, we show in 5637 cells that TNTs mediate the trafficking of RalA protein and transmembrane MHC class III protein leukocyte-specific transcript 1 (LST1). Furthermore, we show that RalGPS2 is essential for nanotubes generation, and stress conditions boost its expression both in 5637 and HEK293 cell lines. Finally, we prove that RalGPS2 interacts with Akt and PDK1, in addition to LST1 and RalA, leading to the formation of a complex that promotes nanotubes formation. In conclusion, our findings suggest that in the tumor microenvironment, RalGPS2 orchestrates the assembly of multimolecular complexes that drive the formation of TNTs.

Squarate Cross-Linked Gelatin Hydrogels as Three-Dimensional Scaffolds for Biomedical Applications.

Hydrogels are useful platforms as three-dimensional (3D) scaffolds for cell culture, drug-release systems, and regenerative medicine applications. Here, we propose a novel chemical cross-linking approach by the use of 3,4-diethoxy-3-cyclobutene-1,2-dione or diethyl squarate for the preparation of 5 and 10% w/v gelatin-based hydrogels. Hydrogels showed good swelling properties, and the 5% gelatin-based hydrogel proved suitable as a 3D cell culture scaffold for the chondrocyte cell line C28/I2. In addition, diffusion properties of different sized molecules inside the hydrogel were determined.

The 3.0 Cell Communication: New Insights in the Usefulness of Tunneling Nanotubes for Glioblastoma Treatment.

Glioblastoma (GBM) is a particularly challenging brain tumor characterized by a heterogeneous, complex, and multicellular microenvironment, which represents a strategic network for treatment escape. Furthermore, the presence of GBM stem cells (GSCs) seems to contribute to GBM recurrence after surgery, and chemo- and/or radiotherapy. In this context, intercellular communication modalities play key roles in driving GBM therapy resistance. The presence of tunneling nanotubes (TNTs), long membranous open-ended channels connecting distant cells, has been observed in several types of cancer, where they emerge to steer a more malignant phenotype. Here, we discuss the current knowledge about the formation of TNTs between different cellular types in the GBM microenvironment and their potential role in tumor progression and recurrence. Particularly, we highlight two prospective strategies targeting TNTs as possible therapeutics: (i) the inhibition of TNT formation and (ii) a boost in drug delivery between cells through these channels. The latter may require future studies to design drug delivery systems that are exchangeable through TNTs, thus allowing for access to distant tumor niches that are involved in tumor immune escape, maintenance of GSC plasticity, and increases in metastatic potential.

Palmitoylethanolamide Modulation of Microglia Activation: Characterization of Mechanisms of Action and Implication for Its Neuroprotective Effects.

Palmitoylethanolamide (PEA) is an endogenous lipid produced on demand by neurons and glial cells that displays neuroprotective properties. It is well known that inflammation and neuronal damage are strictly related processes and that microglia play a pivotal role in their regulation. The aim of the present work was to assess whether PEA could exert its neuroprotective and anti-inflammatory effects through the modulation of microglia reactive phenotypes. In N9 microglial cells, the pre-incubation with PEA blunted the increase of M1 pro-inflammatory markers induced by lipopolysaccharide (LPS), concomitantly increasing those M2 anti-inflammatory markers. Images of microglial cells were processed to obtain a set of morphological parameters that highlighted the ability of PEA to inhibit the LPS-induced M1 polarization and suggested that PEA might induce the anti-inflammatory M2a phenotype. Functionally, PEA prevented Ca2+ transients in both N9 cells and primary microglia and antagonized the neuronal hyperexcitability induced by LPS, as revealed by multi-electrode array (MEA) measurements on primary cortical cultures of neurons, microglia, and astrocyte. Finally, the investigation of the molecular pathway indicated that PEA effects are not mediated by toll-like receptor 4 (TLR4); on the contrary, a partial involvement of cannabinoid type 2 receptor (CB2R) was shown by using a selective receptor inverse agonist.

Synthesis, Molecular Modeling and Biological Evaluation of Metabolically Stable Analogues of the Endogenous Fatty Acid Amide Palmitoylethanolamide.

Palmitoylethanolamide (PEA) belongs to the class of N-acylethanolamine and is an endogenous lipid potentially useful in a wide range of therapeutic areas; products containing PEA are licensed for use in humans as a nutraceutical, a food supplement, or food for medical purposes for its analgesic and anti-inflammatory properties demonstrating efficacy and tolerability. However, the exogenously administered PEA is rapidly inactivated; in this process, fatty acid amide hydrolase (FAAH) plays a key role both in hepatic metabolism and in intracellular degradation. So, the aim of the present study was the design and synthesis of PEA analogues that are more resistant to FAAH-mediated hydrolysis. A small library of PEA analogues was designed and tested by molecular docking and density functional theory calculations to find the more stable analogue. The computational investigation identified RePEA as the best candidate in terms of both synthetic accessibility and metabolic stability to FAAH-mediated hydrolysis. The selected compound was synthesized and assayed ex vivo to monitor FAAH-mediated hydrolysis and to confirm its anti-inflammatory properties. 1H-NMR spectroscopy performed on membrane samples containing FAAH in integral membrane protein demonstrated that RePEA is not processed by FAAH, in contrast with PEA. Moreover, RePEA retains PEA's ability to inhibit LPS-induced cytokine release in both murine N9 microglial cells and human PMA-THP-1 cells.

Serum-deprived differentiated neuroblastoma F-11 cells express functional dorsal root ganglion neuron properties.

The isolation and culture of dorsal root ganglion (DRG) neurons cause adaptive changes in the expression and regulation of ion channels, with consequences on neuronal excitability. Considering that not all neurons survive the isolation and that DRG neurons are heterogeneous, it is difficult to find the cellular subtype of interest. For this reason, researchers opt for DRG-derived immortal cell lines to investigate endogenous properties. The F-11 cell line is a hybridoma of embryonic rat DRG neurons fused with the mouse neuroblastoma line N18TG2. In the proliferative condition, F-11 cells do not display a gene expression profile correspondent with specific subclasses of sensory neurons, but the most significant differences when compared with DRGs are the reduction of voltage-gated sodium, potassium and calcium channels, and the small amounts of TRPV1 transcripts. To investigate if functional properties of mature F-11 cells showed more similarities with those of isolated DRG neurons, we differentiated them by serum deprivation. Potassium and sodium currents significantly increased with differentiation, and biophysical properties of tetrodotoxin (TTX)-sensitive currents were similar to those characterized in small DRG neurons. The analysis of the voltage-dependence of calcium currents demonstrated the lack of low threshold activated components. The exclusive expression of high threshold activated Ca2+ currents and of TTX-sensitive Na+ currents correlated with the generation of a regular tonic electrical activity, which was recorded in the majority of the cells (80%) and was closely related to the activity of afferent TTX-sensitive A fibers of the proximal urethra and the bladder. Responses to capsaicin and substance P were also recorded in ~20% and ~80% of cells, respectively. The percentage of cells responsive to acetylcholine was consistent with the percentage referred for rat DRG primary neurons and cell electrical activity was modified by activation of non-NMDA receptors as for embryonic DRG neurons. These properties and the algesic profile (responses to pH5 and sensitivity to both ATP and capsaicin), proposed in literature to define a sub-classification of acutely dissociated rat DRG neurons, suggest that differentiated F-11 cells express receptors and ion channels that are also present in sensory neurons.

Differential Exchange of Multifunctional Liposomes Between Glioblastoma Cells and Healthy Astrocytes via Tunneling Nanotubes.

Despite advances in cancer therapies, nanomedicine approaches including the treatment of glioblastoma (GBM), the most common, aggressive brain tumor, remains inefficient. These failures are likely attributable to the complex and not yet completely known biology of this tumor, which is responsible for its strong invasiveness, high degree of metastasis, high proliferation potential, and resistance to radiation and chemotherapy. The intimate connection through which the cells communicate between them plays an important role in these biological processes. In this scenario, tunneling nanotubes (TnTs) are recently gaining importance as a key feature in tumor progression and in particular in the re-growth of GBM after surgery. In this context, we firstly identified structural differences of TnTs formed by U87-MG cells, as model of GBM cells, in comparison with those formed by normal human astrocytes (NHA), used as a model of healthy cells. Successively, we have studied the possibility to exploit U87-MG TnTs as drug-delivery channels in cancer therapy, using liposomes composed of cholesterol/sphingomyelin and surface functionalized with mApoE and chlorotoxin peptides (Mf-LIP) as nanovehicle model. The results showed that U87-MG cells formed almost exclusively thick and long protrusions, whereas NHA formed more thin and short TnTs. Considering that thick TnTs are more efficient in transport of vesicles and organelles, we showed that fluorescent-labeled Mf-LIP can be transported via TnTs between U87-MG cells and with less extent through the protrusions formed by NHA cells. Our results demonstrate that nanotubes are potentially useful as drug-delivery channels for cancer therapy, facilitating the intercellular redistribution of this drug in close and far away cells, thus reaching isolated tumor niches that are hardly targeted by simple drug diffusion in the brain parenchyma. Moreover, the differences identified in TnTs formed by GBM and NHA cells can be exploited to increase treatment precision and specificity.

Plant Polyphenols and Exendin-4 Prevent Hyperactivity and TNF-α Release in LPS-Treated In vitro Neuron/Astrocyte/Microglial Networks.

Increasing evidence supports a decisive role for neuroinflammation in the neurodegenerative process of several central nervous system (CNS) disorders. Microglia are essential mediators of neuroinflammation and can regulate a broad spectrum of cellular responses by releasing reactive oxygen intermediates, nitric oxide, proteases, excitatory amino acids, and cytokines. We have recently shown that also in ex-vivo cortical networks of neurons, astrocytes and microglia, an increased level of tumor necrosis factor-alpha (TNF-α) was detected a few hours after exposure to the bacterial endotoxin lipopolysaccharide (LPS). Simultaneously, an atypical "seizure-like" neuronal network activity was recorded by multi-electrode array (MEA) electrophysiology. These effects were prevented by minocycline, an established anti-inflammatory antibiotic. We show here that the same inhibitory effect against LPS-induced neuroinflammation is exerted also by natural plant compounds, polyphenols, such as curcumin (CU, curcuma longa), crocin (CR, saffron), and resveratrol (RE, grape), as well as by the glucagon like peptide-1 receptor (GLP-1R) agonist exendin-4 (EX-4). The drugs tested also caused per-se early transient (variable) changes of network activity. Since it has been reported that LPS-induced neuroinflammation causes rearrangements of glutamate transporters in astrocytes and microglia, we suggest that neural activity could be putatively increased by an imbalance of glial glutamate transporter activity, leading to prolonged synaptic glutamatergic dysregulation.

The deubiquitinating enzyme UBPy/USP8 interacts with TrkA and inhibits neuronal differentiation in PC12 cells.

The tropomyosin-related kinase (Trk) family of receptor tyrosine kinases controls synaptic function, plasticity and sustains differentiation, morphology, and neuronal cell survival. Understanding Trk receptors down-regulation and recycling is a crucial step to point out sympathetic and sensory neuron function and survival. PC12 cells derived from pheochromocytoma of the rat adrenal medulla have been widely used as a model system for studies of neuronal differentiation as they respond to nerve growth factor (NGF) with a dramatic change in phenotype and acquire a number of properties characteristic of sympathetic neurons. In this study we demonstrated that in PC12 cells the TrkA receptor interacts with the deubiquitinating enzyme USP8/UBPy in a NGF-dependent manner and that it is deubiquitinated in vivo and in vitro by USP8. USP8 overexpression blocked NGF-induced neurites outgrowth while the overexpression of the catalytically inactive mutant USP8/UBPy(C748A) caused a marked increase of cell differentiation. Localization and biochemical experiments have point out that USP8 and TrkA partially co-localize in endosomes after NGF stimulation. Finally we have studied the role played by USP8 on TrkA turnover; using specific siRNA for USP8 we found that USP8 knockdown increases TrkA half-life, suggesting that the deubiquitinating activity of USP8 promotes TrkA degradation.