Evidence of Presynaptic Localization and Function of the c-Jun N-Terminal Kinase.

The c-Jun N-terminal kinase (JNK) is part of a stress signalling pathway strongly activated by NMDA-stimulation and involved in synaptic plasticity. Many studies have been focused on the post-synaptic mechanism of JNK action, and less is known about JNK presynaptic localization and its physiological role at this site. Here we examined whether JNK is present at the presynaptic site and its activity after presynaptic NMDA receptors stimulation. By using N-SIM Structured Super Resolution Microscopy as well as biochemical approaches, we demonstrated that presynaptic fractions contained significant amount of JNK protein and its activated form. By means of modelling design, we found that JNK, via the JBD domain, acts as a physiological effector on T-SNARE proteins; then using biochemical approaches we demonstrated the interaction between Syntaxin-1-JNK, Syntaxin-2-JNK, and Snap25-JNK. In addition, taking advance of the specific JNK inhibitor peptide, D-JNKI1, we defined JNK action on the SNARE complex formation. Finally, electrophysiological recordings confirmed the role of JNK in the presynaptic modulation of vesicle release. These data suggest that JNK-dependent phosphorylation of T-SNARE proteins may have an important functional role in synaptic plasticity.

Optimized "in vitro" culture conditions for human rheumatoid arthritis synovial fibroblasts.

The composition of synovial fluid in rheumatoid arthritis (RA) is complex and strongly influences the microenvironment of joints and it is an inseparable element of the disease. Currently, "in vitro" studies are performed on RA cells cultured in the presence of either recombinant proinflammatory cytokines-conditioned medium or medium alone. In this study, we evaluated the use of synovial fluid, derived from RA patients, as optimal culture condition to perform "in vitro" studies on RA synovial fibroblasts. We observed that synovial fluid is more effective in inducing cell proliferation with respect to TNF-alpha or culture medium alone. Spontaneous apoptosis in fibroblasts was also decreased in response to synovial fluid. The expression of proinflammatory cytokines in the presence of synovial fluid was significantly elevated with respect to cells cultured with TNF-alpha or medium, and the overall morphology of cells was also modified. In addition, modulation of intracellular calcium dynamics elicited in response to synovial fluid or TNF-alpha exposure is different and suggests a role for the purinergic signalling in the modulation of the effects. These results emphasize the importance of using RA synovial fluid in "in vitro" studies involving RA cells, in order to reproduce faithfully the physiopathological environmental characteristic of RA joints.

A soluble biocompatible guanidine-containing polyamidoamine as promoter of primary brain cell adhesion and in vitro cell culturing.

This paper reports on a novel application of an amphoteric water-soluble polyamidoamine named AGMA1 bearing 4-butylguanidine pendants. AGMA1 is an amphoteric, prevailingly cationic polyelectrolyte with isoelectric point of about 10. At pH 7.4 it is zwitterionic with an average of 0.55 excess positive charges per unit, notwithstanding it is highly biocompatible. In this work, it was found that AGMA1 surface-adsorbed on cell culturing coverslips exhibits excellent properties as adhesion and proliferation promoter of primary brain cells such as microglia, as well as of hippocampal neurons and astrocytes. Microglia cells cultured on AGMA1-coated coverslips substrate displayed the typical resting, ramified morphology of those cultured on poly-L-lysine and poly-L-ornithine, employed as reference substrates. Mixed cultures of primary astrocytes and neuronal cells grown on AGMA1- and poly-L-lysine coated coverslips were morphologically undistinguishable. On both substrates, neurons differentiated axon and dendrites and eventually established perfectly functional synaptic contacts. Quantitative immunocytochemical staining revealed no difference between AGMA1 and poly-L-lysine. Electrophysiological experiments allowed recording neuron spontaneous activity on AGMA1. In addition, cell cultures on both AGMA1 and PLL displayed comparable excitatory and inhibitory neurotransmission, demonstrating that the synaptic contacts formed were fully functional.

Overflow microfluidic networks: application to the biochemical analysis of brain cell interactions in complex neuroinflammatory scenarios.

Neuroinflammation plays a central role in neurodegenerative diseases and involves a large number of interactions between different brain cell types. Unraveling the complexity of cell-cell interaction in neuroinflammation is crucial for both clarifying the molecular mechanisms involved and increasing efficacy in drug development. Here, we provide a versatile analytical method for specifically addressing cell-to-cell communication, using primary brain cells, a microfluidic device, and a multiparametric readout approach. Different cell types are plated in separate chambers of a microfluidic network so that culturing conditions can be independently controlled and single cell types can be selectively primed with different stimuli. When chambers are microfluidically connected, the specific contribution of each cell type can be finely monitored by analyzing morphology, vitality, calcium dynamics, and electrophysiology parameters. We exemplify this approach by examining the role of astrocytes derived from two different brain regions (cortex and hippocampus) on neuronal viability in two types of neuroinflammatory insults, namely, metabolic stress and exposure to amyloid ß fibrils, and demonstrate regional differences in glial control of neuronal physiopathology. In particular, we show that during metabolic stress, cortical but not hippocampal astrocytes play a neuroprotective role; also, in an exacerbated inflammatory scenario consisting in the exposure to Aß + IL-1ß, hippocampal but not cortical astrocytes play a detrimental role on neurons. Aside from bringing novel insights into the glial role in neuroinflammation, the method presented here represents a promising tool for addressing a wide range of biological and biochemical phenomena, characterized by a complex interaction of multiple cell types.

Overflow microfluidic networks for open and closed cell cultures on chip.

Microfluidics have a huge potential in biomedical research, in particular for studying interactions among cell populations that are involved in complex diseases. Here, we present "overflow" microfluidic networks (oMFNs) for depositing, culturing, and studying cell populations, which are plated in a few microliters of cell suspensions in one or several open cell chambers inside the chip and subsequently cultured for several days in vitro (DIV). After the cells have developed their phenotype, the oMFN is closed with a lid bearing microfluidic connections. The salient features of the chips are (1) overflow zones around the cell chambers for drawing excess liquid by capillarity from the chamber during sealing the oMFN with the lid, (2) flow paths from peripheral pumps to cell chambers and between cell chambers for interactive flow control, (3) transparent cell chambers coated with cell adhesion molecules, and (4) the possibility to remove the lid for staining and visualizing the cells after, for example, fixation. Here, we use a two-chamber oMFN to show the activation of purinergic receptors in microglia grown in one chamber, upon release of adenosine triphosphate (ATP) from astrocytes that are grown in another chamber and challenged with glutamate. These data validate oMFNs as being particularly relevant for studying primary cells and dissecting the specific intercellular pathways involved in neurodegenerative and neuroinflammatory brain diseases.

A microfluidic device for depositing and addressing two cell populations with intercellular population communication capability.

We present a method for depositing cells in the microchambers of a sealed microfluidic device and establishing flow across the chambers independently and serially. The device comprises a transparent poly(dimethylsiloxane) (PDMS) microfluidic network (MFN) having 2 cell chambers with a volume of 0.49 microL, 6 microchannels for servicing the chambers, and 1 microchannel linking both chambers. The MFN is sealed with a Si chip having 6 vias and ports that can be left open or connected to high-precision pumps. Liquids are drawn through each chamber in parallel or sequentially at flow rates from 0.1 to 10 microL min(-1). Plugs of liquid as small as 0.5 microL can be passed in one chamber within 5 s to 5 min. Plugs of liquid can also be introduced into a chamber for residence times of up to 30 min. By injecting different liquids into 3 ports, 3 adjacent laminar streams of liquid can be drawn inside one chamber with lateral concentration gradients between the streams ranging from 20 to 500 microm. The flexibility of this device for depositing cells and exposing them to liquids in parallel or serially is illustrated by depositing two types of cells, murine N9 microglia and human SH-S5Y5 neuroblastoma. Microfluidic communication between the chambers is illustrated by stimulating N9 microglia using ATP to induce these cells to release plasma membrane vesicles. The vesicles are drawn through the second chamber containing neuroblastoma and collected in a port of the device for off-chip analysis using confocal fluorescence microscopy. Cells in the MFN can also be fixed using a solution of formaldehyde for further analysis after disassembly of the MFN and Si lid. This microfluidic device offers a simple, flexible, and powerful method for depositing two cell populations in separate chambers and may help investigating pathways between the cells populations.

Blockade of IGF2R improves muscle regeneration and ameliorates Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a debilitating fatal X-linked muscle disorder. Recent findings indicate that IGFs play a central role in skeletal muscle regeneration and development. Among IGFs, insulinlike growth factor 2 (IGF2) is a key regulator of cell growth, survival, migration and differentiation. The type 2 IGF receptor (IGF2R) modulates circulating and tissue levels of IGF2 by targeting it to lysosomes for degradation. We found that IGF2R and the store-operated Ca2+ channel CD20 share a common hydrophobic binding motif that stabilizes their association. Silencing CD20 decreased myoblast differentiation, whereas blockade of IGF2R increased proliferation and differentiation in myoblasts via the calmodulin/calcineurin/NFAT pathway. Remarkably, anti-IGF2R induced CD20 phosphorylation, leading to the activation of sarcoplasmic/endoplasmic reticulum Ca2+ -ATPase (SERCA) and removal of intracellular Ca2+ . Interestingly, we found that IGF2R expression was increased in dystrophic skeletal muscle of human DMD patients and mdx mice. Blockade of IGF2R by neutralizing antibodies stimulated muscle regeneration, induced force recovery and normalized capillary architecture in dystrophic mdx mice representing an encouraging starting point for the development of new biological therapies for DMD.

Anti-inflammatory Effect of Somatostatin Analogue Octreotide on Rheumatoid Arthritis Synoviocytes.

Somatostatin and its analogues are known to have modulatory effects on immune response and their anti-proliferative, anti-angiogenic, and analgesic properties make them attractive candidates for a therapeutic use in immune-mediated diseases, such as rheumatoid arthritis. Here, we demonstrate the ability of the somatostatin analogue octreotide to inhibit interleukin-15 and to increase interleukin-10 production by rheumatoid arthritis fibroblast-like synovial cells maintained in a chronic inflammatory state. We also prove that the inhibitory effect of octreotide on interleukin-15 and tumor necrosis factor-α production depended on the increase in interleukin-10, since neutralizing anti-interleukin-10 antibody was able to partially reverse this inhibition. In addition, our observations suggest an octreotide control on purinergic signaling, with an inhibitory effect on purinergic P2X and P2Y receptors activation. This would have great implications, considering the roles of P2 receptors in the onset of inflammation. Data here reported extend knowledge on the biological action of octreotide and underline its multiple effects on immune response, which could make octreotide an attractive and valid support for the therapy of diseases where several inflammatory mediators are involved, such as rheumatoid arthritis, and in which the simultaneous action on different aspects can be a successful strategy.

Characterization of a Monoclonal Antibody Specific for the Growth Hormone Secretagogue Receptor.

Ghrelin is an orexigenic peptide hormone that primarily regulates growth hormone secretion, food intake, and energy homeostasis. It has been shown to also play a role in numerous higher brain functions, such as the regulation of inflammation and cell proliferation. Ghrelin is the endogenous ligand of the growth hormone secretagogue receptor (GHSR), a G-protein-coupled receptor highly expressed in brain and detectable in some peripheral tissues. The wide distribution of ghrelin receptor and the number of tissues and cell types known to respond to ghrelin suggest that a number of systems may be affected by treatment with this hormone or its analogues. In this study, we characterized a new GHSR specific monoclonal antibody recognizing specifically the ghrelin receptor. This could be a useful tool for immunoassays aimed at obtaining insights into the physiological and pathological significance of the GHSR/ghrelin system.

A functional role of postsynaptic density-95-guanylate kinase-associated protein complex in regulating Shank assembly and stability to synapses.

Postsynaptic density (PSD) proteins include scaffold, cytoskeletal, and signaling proteins that structurally and functionally interact with glutamate receptors and other postsynaptic membrane proteins. The molecular mechanisms regulating the assembly of PSD proteins and their associations with synapses are still widely unknown. We investigated the molecular mechanisms of Shank1 targeting and synapse assembly by looking at the function of guanylate kinase-associated protein (GKAP) and PSD-95 interactions. Shank1 when it is not associated to GKAP, which binds to the Shank PSD-95-Discs Large-zona occludens-1 domain, forms filamentous and fusiform structures in which the Src homology 3 domain specifically interacts with the ankyrin repeat domain, thus allowing its multimerization via a novel form of intermolecular interaction. Surprisingly, in both COS-7 cells and hippocampal neurons, GKAP forms insoluble aggregates with Shank that colocalize with heat shock protein 70 and neurofilaments, two markers of the aggresomes in which misfolded proteins accumulate. However, the two proteins are organized in clusters in COS cells and synaptic clusters in neurons when both are overexpressed and associated with wild-type PSD-95, but not with palmitoylation-deficient PSD-95. Synaptic activity in neurons induces the formation of Shank and GKAP intracellular aggregation and degradation. Similarly, the overexpression of a GKAP mutant that is incapable of binding PSD-95 induces Shank aggregation and degradation in neurons. Our data suggest a possible functional and structural role of the PSD-95-GKAP complex in Shank and PSD protein assembly and stability to synapses.

Mesenchymal stem cell differentiation on electrochemically modified titanium: an optimized approach for biomedical applications.

PURPOSE: To speed up the osteointegration process, surface-treated titanium has been widely used in dental and orthopedic applications. The present work describes a new silicon-based anodic spark deposition (ASD) treatment and investigates the properties of the surfaces obtained, focusing on their capability to modulate the osteogenic differentiation potential of adult mesenchymal stem cells (MSCs). METHODS: The surfaces examined were obtained from commercially pure grade 2 titanium by a single-step ASD (SUM) eventually followed by a thermal treatment in alkali solution (SUMNa), while acid-etched titanium (AE; NextMaterials s.r.l.) was selected as a control. Their morphology, elemental composition, crystallographic structure of the Ti2O layer, wettability and topography were evaluated by scanning electron microscopy, energy-dispersive X-ray spectroscopy, thin-film X-ray diffraction, contact angle measurements and laser profilometry, respectively. MSCs' response to surface properties was assessed by examining cell morphology and viability by scanning electron microscopy and Alamar Blue assay®, while their osteogenic differentiation potential was investigated by evaluating the levels of the enzyme alkaline phosphatase (ALP) and the degree of calcium accumulation by Alizarin Red-S (AR-S) staining. RESULTS: The proposed ASD treatment has allowed the obtaining of surfaces with round-shaped micrometric pores, enriched in calcium, phosphorus and silicon and significantly more wettable than controls; furthermore, the treatment has been shown to promote MSC proliferation and the degree of in vitro mineralization. CONCLUSIONS: The described ASD treatment may be an effective technique to modify the surface cues of titanium implants, aiming at enhancing the conveying of osteoprogenitor cells and their functional differentiation in bone cells.

Pharmacology on microfluidics: multimodal analysis for studying cell-cell interaction.

Understanding the mechanisms of cell-cell interaction is a key unanswered question in modern pharmacology, given crosstalk defects are at the basis of many pathologies. Microfluidics represents a valuable tool for analyzing intercellular communication mediated by transmission of soluble signals, as occurring for example between neurons and glial cells in neuroinflammation, or between tumor and surrounding cells in cancer. However, the use of microfluidics for studying cell behavior still encompasses many technical and biological challenges. In this review, a state of the art of successes, potentials and limitations of microfluidics applied to key biological questions in modern pharmacology is analyzed and commented.

A simple method to generate adipose stem cell-derived neurons for screening purposes.

Strategies involved in mesenchymal stem cell (MSC) differentiation toward neuronal cells for screening purposes are characterized by quality and quantity issues. Differentiated cells are often scarce with respect to starting undifferentiated population, and the differentiation process is usually quite long, with high risk of contamination and low yield efficiency. Here, we describe a novel simple method to induce direct differentiation of MSCs into neuronal cells, without neurosphere formation. Differentiated cells are characterized by clear morphological changes, expression of neuronal specific markers, showing functional response to depolarizing stimuli and electrophysiological properties similar to those of developing neurons. The method described here represents a valuable tool for future strategies aimed at personalized screening of therapeutic agents in vitro.

The expression of GHS-R in primary neurons is dependent upon maturation stage and regional localization.

Ghrelin is a hormone with a crucial role in the regulation of appetite, regulation of inflammation, glucose metabolism and cell proliferation. In the brain ghrelin neurons are located in the cortex (sensorimotor area, cingular gyrus), and the fibres of ghrelin neurons in hypothalamus project directly to the dorsal vagal complex (DVC). Ghrelin binds the growth hormone secretagogue receptor (GHS-R) a G-protein-coupled receptor with a widespread tissue distribution, indeed these receptors are localized both in nonnervous, organs/tissues (i.e. adipose tissue, myocardium, adrenals, gonads, lung, liver, arteries, stomach, pancreas, thyroid, and kidney) as well as in central nervous system (CNS) and higher levels of expression in the pituitary gland and the hypothalamus and lower levels of expression in other organs, including brain. A GHS-R specific monoclonal antibody has been developed and characterized and through it we demonstrate that GHS-R is expressed in primary neurons and that its expression is dependent upon their developmental stage and shows differences according to the brain region involved, with a more pronounced expression in hippocampal rather than cortical neurons. A characterization of GHS-R within the central nervous system is of extreme importance in order to gain insights on its role in the modulation of neurodegenerative events such as Alzheimer's disease.

Expression of CD20 reveals a new store-operated calcium entry modulator in skeletal muscle.

Among the scarce available data about the biological role of the membrane protein CD20, there is some evidence that this protein functions as a store-operated Ca(2+) channel and/or regulates transmembrane Ca(2+) trafficking. Recent findings indicate that store-operated Ca(2+) entry (SOCE) plays a central role in skeletal muscle function and development, but there remain a number of unresolved issues relating to SOCE modulation in this tissue. Here we describe CD20 expression in skeletal muscle, verifying its membrane localization in myoblasts and adult muscle fibers. Additionally, we show that inhibition of CD20 through antibody binding or gene silencing resulted in specific impairment of SOCE in C2C12 myoblasts. Our results provide novel insights into the CD20 expression pattern, and suggest that functional CD20 is required for SOCE to consistently occur in C2C12 myoblasts. These findings may contribute to future identification of mechanisms and molecules involved in the fine regulation of store-operated Ca(2+) entry in skeletal muscle.

Proteomic analysis of activity-dependent synaptic plasticity in hippocampal neurons.

Following long-term treatment with bicuculline and tetrodotoxin (TTX) aimed at modifying synaptic activity in cultured neurons, we used a proteomic approach to identify the associated changes in protein expression. The neurons were left untreated, or treated with bicuculline or TTX, and fractionated by means of differential detergent extraction, after which the proteins in each fraction were separated by means of two-dimensional (2D) gel electrophoresis, and 57 proteins of interest were identified by mass spectrometry. The proteins that showed altered expression and/or post-translational modifications include proteins or enzymes involved in regulating cell and protein metabolism, the cytoskeleton, or mitochondrial activity. These results suggest that extensive alterations in neuronal protein expression take place as a result of increased or decreased synaptic activity.

DNA methylation regulates tissue-specific expression of Shank3.

Tissue-specific gene expression can be controlled by epigenetic modifications such as DNA methylation. SHANK3, together with its homologues SHANK1 and SHANK2, has a central functional and structural role in excitatory synapses and is involved in the human chromosome 22q13 deletion syndrome. In this report, we show by DNA methylation analysis in lymphocytes, brain cortex, cerebellum and heart that the three SHANK genes possess several methylated CpG boxes, but only SHANK3 CpG islands are highly methylated in tissues where protein expression is low or absent and unmethylated where expression is present. SHANK3 protein expression is significantly reduced in hippocampal neurons after treatment with methionine, while HeLa cells become able to express SHANK3 after treatment with 5-Aza-2'-deoxycytidine. Altogether, these data suggest the existence of a specific epigenetic control mechanism regulating SHANK3, but not SHANK1 and SHANK2, expression.