Transcriptional repression of the oncofetal LIN28B gene by the transcription factor SOX6.

The identification of regulatory networks contributing to fetal/adult gene expression switches is a major challenge in developmental biology and key to understand the aberrant proliferation of cancer cells, which often reactivate fetal oncogenes. One key example is represented by the developmental gene LIN28B, whose aberrant reactivation in adult tissues promotes tumor initiation and progression. Despite the prominent role of LIN28B in development and cancer, the mechanisms of its transcriptional regulation are largely unknown. Here, by using quantitative RT-PCR and single cell RNA sequencing data, we show that in erythropoiesis the expression of the transcription factor SOX6 matched a sharp decline of LIN28B mRNA during human embryo/fetal to adult globin switching. SOX6 overexpression repressed LIN28B not only in a panel of fetal-like erythroid cells (K562, HEL and HUDEP1; ≈92% p < 0.0001, 54% p = 0.0009 and ≈60% p < 0.0001 reduction, respectively), but also in hepatoblastoma HepG2 and neuroblastoma SH-SY5H cells (≈99% p < 0.0001 and ≈59% p < 0.0001 reduction, respectively). SOX6-mediated repression caused downregulation of the LIN28B/Let-7 targets, including MYC and IGF2BP1, and rapidly blocks cell proliferation. Mechanistically, Lin28B repression is accompanied by SOX6 physical binding within its locus, suggesting a direct mechanism of LIN28B downregulation that might contribute to the fetal/adult erythropoietic transition and restrict cancer proliferation.

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.

Prussian Blue Nanoparticle-Mediated Scalable Thermal Stimulation for In Vitro Neuronal Differentiation.

Heating has recently been applied as an alternative to electrical stimulation to modulate excitability and to induce neuritogenesis and the expression of neuronal markers; however, a long-term functional differentiation has not been described so far. Here, we present the results obtained by a new approach for scalable thermal stimulation on the behavior of a model of dorsal root ganglion neurons, the F-11 cell line. Initially, we performed experiments of bulk stimulation in an incubator for different time intervals and temperatures, and significant differences in neurite elongation and in electrophysiological properties were observed in cultures exposed at 41.5 °C for 30 min. Thus, we exposed the cultures to the same temperature increase using a near-infrared laser to irradiate a disc of Prussian blue nanoparticles and poly-vinyl alcohol that we had adhered to the outer surface of the petri dish. In irradiated cells, neurites were significantly longer, and the electrophysiological properties (action potential firing frequency and spontaneous activity) were significantly increased compared to the control. These results show for the first time that a targeted thermal stimulation could induce morphological and functional neuronal differentiation and support the future application of this method as a strategy to modify neuronal behavior in vivo.

Role of Nuclear Receptors in Controlling Erythropoiesis.

Nuclear receptors (NRs), are a wide family of ligand-regulated transcription factors sharing a common modular structure composed by an N-terminal domain and a ligand-binding domain connected by a short hinge linker to a DNA-binding domain. NRs are involved in many physiological processes, including metabolism, reproduction and development. Most of them respond to small lipophilic ligands, such as steroids, retinoids, and phospholipids, which act as conformational switches. Some NRs are still "orphan" and the search for their ligands is still ongoing. Upon DNA binding, NRs can act both as transcriptional activators or repressors of their target genes. Theoretically, the possibility to modulate NRs activity with small molecules makes them ideal therapeutic targets, although the complexity of their signaling makes drug design challenging. In this review, we discuss the role of NRs in erythropoiesis, in both homeostatic and stress conditions. This knowledge is important in view of modulating red blood cells production in disease conditions, such as anemias, and for the expansion of erythroid cells in culture for research purposes and for reaching the long-term goal of cultured blood for transfusion.

Neoglycosylated Collagen: Effect on Neuroblastoma F-11 Cell Lines.

The regeneration of the nervous system is a challenging task. Currently, regenerative medicine approaches that exploit nature-inspired cues are being studied and hold great promise. The possibility to use protein-based matrices functionalized with small oligo- and monosaccharides is of interest since these can be finely tuned to better mimic the native environment. Collagen has been selected as a promising material that has the potential to be further tailored to incorporate carbohydrates in order to drive cell behavior towards neuroregeneration. Indeed, the grafting of carbohydrates to collagen 2D matrices is proved to enhance its biological significance. In the present study, collagen 2D matrices were grafted with different carbohydrate epitopes, and their potential to drive F-11 neuroblastoma cells towards neuronal differentiation was evaluated. Collagen functionalized with α-glucosides was able to differentiate neuroblastoma cells into functional neurons, while sialyl α-(2→6)-galactosides stimulated cell proliferation.

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.

Multifunctionalized hydrogels foster hNSC maturation in 3D cultures and neural regeneration in spinal cord injuries.

Three-dimensional cell cultures are leading the way to the fabrication of tissue-like constructs useful to developmental biology and pharmaceutical screenings. However, their reproducibility and translational potential have been limited by biomaterial and culture media compositions, as well as cellular sources. We developed a construct comprising synthetic multifunctionalized hydrogels, serum-free media, and densely seeded good manufacturing practice protocol-grade human neural stem cells (hNSC). We tracked hNSC proliferation, differentiation, and maturation into GABAergic, glutamatergic, and cholinergic neurons, showing entangled electrically active neural networks. The neuroregenerative potential of the "engineered tissue" was assessed in spinal cord injuries, where hNSC-derived progenitors and predifferentiated hNSC progeny, embedded in multifunctionalized hydrogels, were implanted. All implants decreased astrogliosis and lowered the immune response, but scaffolds with predifferentiated hNSCs showed higher percentages of neuronal markers, better hNSC engraftment, and improved behavioral recovery. Our hNSC-construct enables the formation of 3D functional neuronal networks in vitro, allowing novel strategies for hNSC therapies in vivo.

Lactoferrin-loaded contact lenses counteract cytotoxicity caused in vitro by keratoconic tears.

Lactoferrin (LF), an iron-binding protein with antioxidant activity, is significantly reduced in the lacrimal film of patients affected by keratoconus (KC) compared to healthy subjects, and this is supposed to be the cause for tear iron increase and consequent iron deposition and oxygen free radical accumulation in the cornea. We decided to study if LF-loaded contact lenses (LF-CLs) could exert antioxidant activity on epithelial cells incubated in tears collected from two patients affected by keratoconus (KC1 and KC2). Moreover through this model we indirectly estimated iron concentration in the tears of healthy and KC subjects. Reflex tears were collected during the first 2 min of light or onion-induced lacrimation and stored at -20 °C. After incubation in tears for 18 h, mortality of epithelial cells was investigated by trypan blue exclusion test. Successively, LF-CLs were deposed on cells incubated in KC1 tears. For the indirect determination of iron content, cells were incubated with LF 1.2 mg/mL and different FeSO4 concentrations, and for the estimation of iron from the patient's tears, cells were incubated with free serum medium and healthy tears (1:1) and different FeSO4 concentrations. Epithelial cells incubated with reflex tears of KC patients showed increased mortality (27.7 ± 3.9%, p = 0.0003, for KC1 and 17.6 ± 0.95%, p = 0.014, for KC2) compared to epithelial cells maintained in control healthy tears (8.6 ± 1.2%). This difference in mortality was correlated with tear iron concentration, which was estimated at 4.58 µg/mL for the healthy subjects, at 56.28 µg/mL for KC1, and at 8.7 µg/mL for KC2 patient. Application of LF-CLs counteracted KC tear cytotoxicity restoring viability obtained in the presence of control tears. Therapeutic contact lenses obtained by LF loading can reduce oxidative stress induced by patients' tears and might represent an efficient device to arrest the progression of keratoconus.

Lactoferrin-loaded contact lenses: eye protection against oxidative stress.

PURPOSE: Tear fluid contains antioxidative compounds, vitamin C, glutathione, superoxide dismutase, and lactoferrin (LF), which protect the corneal epithelium from the effects of ultraviolet irradiation, direct airflow, and chemical agents. However, these natural defenses against oxidative stress can decrease, favoring the development of anterior eye disorders, such as keratoconus, dry eye, and Sjögren syndrome. LF is an iron-binding glycoprotein, present in mammalian secretions such as tears and milk, endowed with different physiological functions such as antimicrobial, antiviral, and antioxidant activities. In this work, we studied the capability of different soft contact lenses to adsorb and release LF to restore cellular viability in oxidative stress conditions. METHODS: Three types of contact lenses (filcon V, galyfilcon A, and filcon IB) were loaded with LF and then incubated with TsA or human corneal epithelial primary cells. After oxidative stress induction with 250 µM or 125 µM H2O2, cell viability was evaluated. RESULTS: Data showed that the highest quantity of LF loaded in contact lenses was between 61 µg (for filcon V) and 39 µg (for filcon IB); the release was between 49% and 100% of protein adsorbed. LF released from contact lenses maintained its antioxidant activity at least for 24 hours and was able to protect human epithelial cells from the detrimental effects of oxidative stress. CONCLUSIONS: These results demonstrate that LF-loaded contact lenses could represent a new therapeutic approach to treat ocular surface pathologies characterized by high levels of oxidative stress.

Hydrogen peroxide mechanosynthesis in siloxane-hydrogel contact lenses.

Drug-loaded contact lenses are emerging as the preferred treatment method for several ocular diseases, and efforts are being directed to promote extended and controlled delivery. One strategy is based on delivery induced by environmental triggers. One of these triggers can be hydrogen peroxide, since many platforms based on drug-loaded nanoparticles were demonstrated to be hydrogen-peroxide responsive. This is particularly interesting when hydrogen peroxide is the result of a specific pathophysiological condition. Otherwise, an alternative route to induce drug delivery is here proposed, namely the mechano-synthesis. The present work represents the proof-of-concept of the mechanosynthesis of hydrogen peroxide in siloxane-hydrogel contact lenses as a consequence of the cleavage of siloxane bonds at the interface between the polymer and water in aqueous phase. Their spongy morphology makes contact lenses promising systems for mechanical-to-chemical energy conversion, since the amount of hydrogen peroxide is expected to scale with the interfacial area between the polymer and water. The eyelid pressure during wear is sufficient to induce the hydrogen peroxide synthesis with concentrations which are biocompatible and suitable to trigger the drug release through hydrogen-peroxide-responsive platforms. For possible delivery on demand, the integration of piezoelectric polymers in the siloxane-hydrogel contact lenses could be designed, whose mechanical deformation could be induced by an applied wireless-controlled voltage.

Epigallocatechin-3-gallate and tetracycline differently affect ataxin-3 fibrillogenesis and reduce toxicity in spinocerebellar ataxia type 3 model.

The polyglutamine (polyQ)-containing protein ataxin-3 (AT3) triggers the neurodegenerative disease spinocerebellar ataxia type 3 (SCA3) when its polyQ tract is expanded beyond a critical length. This results in protein aggregation and generation of toxic oligomers and fibrils. Currently, no effective treatment is available for such and other polyQ diseases. Therefore, plenty of investigations are being carried on to assess the mechanism of action and the therapeutic potential of anti-amyloid agents. The polyphenol compound epigallocatechin-3-gallate (EGCG) and tetracycline have been shown to exert some effect in preventing fibrillogenesis of amyloidogenic proteins. Here, we have incubated an expanded AT3 variant with either compound to assess their effects on the aggregation pattern. The process was monitored by atomic force microscopy and Fourier transform infrared spectroscopy. Whereas in the absence of any treatment, AT3 gives rise to amyloid ß-rich fibrils, whose hallmark is the typical glutamine side-chain hydrogen bonding, when incubated in the presence of EGCG it generated soluble, SDS-resistant aggregates, much poorer in ß-sheets and devoid of any ordered side-chain hydrogen bonding. These are off-pathway species that persist until the latest incubation time and are virtually absent in the control sample. In contrast, tetracycline did not produce major alterations in the structural features of the aggregated species compared with the control, but substantially increased their solubility. Both compounds significantly reduced toxicity, as shown by the MTT assay in COS-7 cell line and in a transgenic Caenorhabditis elegans strain expressing in the nervous system an AT3 expanded variant in fusion with GFP.

Neoglucosylated collagen matrices drive neuronal cells to differentiate.

Despite the relevance of carbohydrates as cues in eliciting specific biological responses, glycans have been rarely exploited in the study of neuronal physiology. We report thereby the study of the effect of neoglucosylated collagen matrices on neuroblastoma F11 cell line behavior. Morphological and functional analysis clearly showed that neoglucosylated collagen matrices were able to drive cells to differentiate. These data show for the first time that F11 cells can be driven from proliferation to differentiation without the use of chemical differentiating agents. Our work may offer to cell biologists new opportunities to study neuronal cell differentiation mechanisms in a cell environment closer to physiological conditions.

Ethidium bromide as a marker of mtDNA replication in living cells.

Mitochondrial DNA (mtDNA) in tumor cells was found to play an important role in maintaining the malignant phenotype. Using laser scanning confocal fluorescence microscopy (LSCFM) in a recent work, we reported a variable fluorescence intensity of ethidium bromide (EB) in mitochondria nucleoids of living carcinoma cells. Since when EB is bound to nucleic acids its fluorescence is intensified; a higher EB fluorescence intensity could reflect a higher DNA accessibility to EB, suggesting a higher mtDNA replication activity. To prove this hypothesis, in the present work we studied, by LSCFM, the EB fluorescence in mitochondria nucleoids of living neuroblastoma cells, a model system in which differentiation affects the level of mtDNA replication. A drastic decrease of fluorescence was observed after differentiation. To correlate EB fluorescence intensity to the mtDNA replication state, we evaluated the mtDNA nascent strands content by ligation-mediated real-time PCR, and we found a halved amount of replicating mtDNA molecules in differentiating cells. A similar result was obtained by BrdU incorporation. These results indicate that the low EB fluorescence of nucleoids in differentiated cells is correlated to a low content of replicating mtDNA, suggesting that EB may be used as a marker of mtDNA replication in living cells.

Proteomic and biochemical analyses unveil tight interaction of ataxin-3 with tubulin.

Ataxin-3 consists of an N-terminal globular Josephin domain and an unstructured C-terminal region containing a stretch of consecutive glutamines that triggers an inherited neurodegenerative disorder, spinocerebellar ataxia type 3, when its length exceeds a critical threshold. The pathology results from protein misfolding and intracellular accumulation of fibrillar amyloid-like aggregates. Plenty of work has been carried out to elucidate the protein's physiological role(s), which has shown that ataxin-3 is multifunctional; it acts as a transcriptional repressor, and also has polyubiquitin-binding/ubiquitin-hydrolase activity. In addition, a recent report shows that it participates in sorting misfolded protein to aggresomes, close to the microtubule-organizing center. Since a thorough understanding of the protein's physiological role(s) requires the identification of all the molecular partners interacting with ataxin-3, we pursued this goal by taking advantage of two-dimensional chromatography coupled to tandem mass spectrometry. We found that different ataxin-3 constructs, including the sole Josephin domain, bound alpha- and beta-tubulin from soluble rat brain extracts. Coimmunoprecipitation experiments confirmed this interaction. Also, normal ataxin-3 overexpressed in COS7 cultured cells partially colocalized with microtubules, whereas an expanded variant only occasionally did so, probably due to aggregation. Furthermore, by surface plasmon resonance we determined a dissociation constant of 50-70nM between ataxin-3 and tubulin dimer, which strongly supports the hypothesis of a direct interaction of this protein with microtubules in vivo. These findings suggest an involvement of ataxin-3 in directing aggregated protein to aggresomes, and shed light on the mode of interaction among the different molecular partners participating in the process.

Study of subcellular localization and proteolysis of ataxin-3.

In this work we investigate subcellular localization and proteolytic cleavage of different forms of ataxin-3 (AT-3), the protein responsible for spinocerebellar ataxia type 3. Normal (AT-3Q6 and AT-3Q26) and pathological (AT-3Q72) ataxins-3, as well as two truncated forms lacking poly-Q, were studied. Full-length proteins were also expressed as C14A mutants, in order to assess whether AT-3 autoproteolytic activity was involved in its fragmentation. We found that both normal and pathological proteins localized in the cytoplasm and in the nucleus, as expected, but also in the mitochondria. Microsequencing showed that all ataxins-3 underwent the same proteolytic cleavage, removing the first 27 amino acids. Interestingly, while normal ataxins were further cleaved at a number of caspase sites, pathological AT-3 was proteolyzed to a much lesser extent. This may play a role in the pathogenesis, hampering degradation of aggregation-prone expanded AT-3. In addition, autolytic cleavage was apparently not involved in AT-3 proteolysis.