Cholangiocarcinoma Malignant Traits Are Promoted by Schwann Cells through TGFß Signaling in a Model of Perineural Invasion.

The term cholangiocarcinoma (CCA) defines a class of epithelial malignancies originating from bile ducts. Although it has been demonstrated that CCA patients with perineural invasion (PNI) have a worse prognosis, the biological features of this phenomenon are yet unclear. Our data show that in human intrahepatic CCA specimens with documented PNI, nerve-infiltrating CCA cells display positivity of the epithelial marker cytokeratin 7, lower with respect to the rest of the tumor mass. In an in vitro 3D model, CCA cells move towards a peripheral nerve explant allowing contact with Schwann cells (SCs) emerging from the nerve. Here, we show that SCs produce soluble factors that favor the migration, invasion, survival and proliferation of CCA cells in vitro. This effect is accompanied by a cadherin switch, suggestive of an epithelial-mesenchymal transition. The influence of SCs in promoting the ability of CCA cells to migrate and invade the extracellular matrix is hampered by a specific TGFß receptor 1 (TGFBR1) antagonist. Differential proteomic data indicate that the exposure of CCA cells to SC secreted factors induces the upregulation of key oncogenes and the concomitant downregulation of some tumor suppressors. Taken together, these data concur in identifying SCs as possible promoters of a more aggressive CCA phenotype, ascribing a central role to TGFß signaling in regulating this process.

PAR1 activation affects the neurotrophic properties of Schwann cells.

Protease-activated receptor-1 (PAR1) is the prototypic member of a family of four G-protein-coupled receptors that signal in response to extracellular proteases. In the peripheral nervous system, the expression and/or the role of PARs are still poorly investigated. High PAR1 mRNA expression was found in the rat dorsal root ganglia and the signal intensity of PAR1 mRNA increased in response to sciatic nerve transection. In the sciatic nerve, functional PAR1 receptor was reported at the level of non-compacted Schwann cell myelin microvilli of the nodes of Ranvier. Schwann cells are the principal population of glial cells of the peripheral nervous system which myelinate axons playing an important role during axonal regeneration and remyelination. The present study was undertaken in order to determine if the activation of PAR1 affects the neurotrophic properties of Schwann cells. Our results suggest that the stimulation of PAR1 could potentiate the Schwann cell ability to favour nerve regeneration. In fact, the conditioned medium obtained from Schwann cell cultures challenged with a specific PAR1 activating peptide (PAR1 AP) displays increased neuroprotective and neurotrophic properties with respect to the culture medium from untreated Schwann cells. The proteomic analysis of secreted proteins in untreated and PAR1 AP-treated Schwann cells allowed the identification of factors differentially expressed in the two samples. Some of them (such as macrophage migration inhibitory factor, matrix metalloproteinase-2, decorin, syndecan 4, complement C1r subcomponent, angiogenic factor with G patch and FHA domains 1) appear to be transcriptionally regulated after PAR1 AP treatment as shown by RT-PCR.

Lithium limits trimethyltin-induced cytotoxicity and proinflammatory response in microglia without affecting the concurrent autophagy impairment.

Trimethyltin (TMT) is a highly toxic molecule present as an environmental contaminant causing neurodegeneration particularly of the limbic system both in humans and in rodents. We recently described the occurrence of impairment in the late stages of autophagy in TMT-intoxicated astrocytes. Here we show that similarly to astrocytes also in microglia, TMT induces the precocious block of autophagy indicated by the accumulation of the autophagosome marker, microtubule associated protein light chain 3. Consistent with autophagy impairment we observe in TMT-treated microglia the accumulation of p62/SQSTM1, a protein specifically degraded through this pathway. Lithium has been proved effective in limiting neurodegenerations and, in particular, in ameliorating symptoms of TMT intoxication in rodents. In our in vitro model, lithium displays a pro-survival and anti-inflammatory action reducing both cell death and the proinflammatory response of TMT-treated microglia. In particular, lithium exerts these activities without reducing TMT-induced accumulation of light chain 3 protein. In fact, the autophagic block imposed by TMT is unaffected by lithium administration. These results are of interest as defects in the execution of autophagy are frequently observed in neurodegenerative diseases and lithium is considered a promising therapeutic agent for these pathologies. Thus, it is relevant that this cation can still maintain its pro-survival and anti-inflammatory role in conditions of autophagy block. Copyright © 2016 John Wiley & Sons, Ltd.

Assessment of marine ecosystem services indicators: Experiences and lessons learned from 14 European case studies.

This article shares the experiences, observations, and discussions that occurred during the completing of an ecosystem services (ES) indicator framework to be used at European Union (EU) and Member States' level. The experience base was drawn from 3 European research projects and 14 associated case study sites that include 13 transitional-water bodies (specifically 8 coastal lagoons, 4 riverine estuaries, and 1 fjord) and 1 coastal-water ecosystem. The ES pertinent to each case study site were identified along with indicators of these ES and data sources that could be used for mapping. During the process, several questions and uncertainties arose, followed by discussion, leading to these main lessons learned: 1) ES identification: Some ES that do not seem important at the European scale emerge as relevant at regional or local scales; 2) ES indicators: When direct indicators are not available, proxies for indicators (indirect indicators) might be used, including combined data on monitoring requirements imposed by EU legislation and international agreements; 3) ES mapping: Boundaries and appropriate data spatial resolution must be established because ES can be mapped at different temporal and spatial scales. We also acknowledge that mapping and assessment of ES supports the dialogue between human well-being and ecological status. From an evidence-based marine planning-process point of view, mapping and assessment of marine ES are of paramount importance to sustainable use of marine natural capital and to halt the loss of marine biodiversity. Integr Environ Assess Manag 2016;12:726-734. © 2016 SETAC.

Impairment of the autophagic flux in astrocytes intoxicated by trimethyltin.

Autophagy is a lysosomal catabolic route for protein aggregates and damaged organelles which in different stress conditions, such as starvation, generally improves cell survival. An impairment of this degradation pathway has been reported to occur in many neurodegenerative processes. Trimethyltin (TMT) is a potent neurotoxin present as an environmental contaminant causing tremors, seizures and learning impairment in intoxicated subjects. The present data show that in rat primary astrocytes autophagic vesicles (AVs) appeared after few hours of TMT treatment. The analysis of the autophagic flux in TMT-treated astrocytes was consistent with a block of the late stages of autophagy and was accompanied by a progressive accumulation of the microtubule associated protein light chain 3 (LC3) and of p62/SQSTM1. Interestingly, an increased immunoreactivity for p62/SQSTM1 was also observed in hippocampal astrocytes detected in brain slices of TMT-intoxicated rats. The time-lapse recordings of AVs in EGFP-mCherry-LC3B transfected astrocytes demonstrated a reduced mobility of autophagosomes after TMT exposure respect to control cells. The observed block of the autophagic flux cannot be overcome by known autophagy inducers such as rapamycin or 0.5mM lithium. Although ineffective when used at 0.5mM, lithium at higher concentrations (2mM) was able to protect astrocyte cultures from TMT toxicity. This effect correlated well with its ability to determine the phosphorylation/inactivation of glycogen kinase synthase-3ß (GSK-3ß).

Lithium improves survival of PC12 pheochromocytoma cells in high-density cultures and after exposure to toxic compounds.

Autophagy is an evolutionary conserved mechanism that allows for the degradation of long-lived proteins and entire organelles which are driven to lysosomes for digestion. Different kinds of stressful conditions such as starvation are able to induce autophagy. Lithium and rapamycin are potent autophagy inducers with different molecular targets. Lithium stimulates autophagy by decreasing the intracellular myo-inositol-1,4,5-triphosphate levels, while rapamycin acts through the inhibition of the mammalian target of rapamycin (mTOR). The correlation between autophagy and cell death is still a matter of debate especially in transformed cells. In fact, the execution of autophagy can protect cells from death by promptly removing damaged organelles such as mitochondria. Nevertheless, an excessive use of the autophagic machinery can drive cells to death via a sort of self-cannibalism. Our data show that lithium (used within its therapeutic window) stimulates the overgrowth of the rat Pheochromocytoma cell line PC12. Besides, lithium and rapamycin protect PC12 cells from toxic compounds such as thapsigargin and trimethyltin. Taken together these data indicate that pharmacological activation of autophagy allows for the survival of Pheochromocytoma cells in stressful conditions such as high-density cultures and exposure to toxins.

The enol tautomer of indole-3-pyruvic acid as a biological switch in stress responses.

Indole-3-pyruvic acid (IPA) undergoes in solutions to the keto-enol tautomerism, which appears responsible of its pharmacological effects, as only the enol tautomer is an easy target for oxygen free-radicals and can be transformed directly to kynurenic acid (KYNA). Contrary to expectations, the IPA enol tautomer is rather stable in mammalian tissues, due to the presence of specific tautomerases, favouring the formation of KYNA in the presence of free-radicals. Because of the synergistic effects between glucocorticoids, free-radicals and excitatory aminoacids in chronic stress, the enol tautomer of IPA and KYNA are proposed as physiological metabolites produced in order to shut-off the chronic stress cycle.