Tumoral Neuroligin 1 Promotes Cancer-Nerve Interactions and Synergizes with the Glial Cell Line-Derived Neurotrophic Factor.

Many nervous proteins are expressed in cancer cells. In this report, we asked whether the synaptic protein neuroligin 1 (NLGN1) was expressed by prostatic and pancreatic carcinomas; in addition, given the tendency of these tumors to interact with nerves, we asked whether NLGN1 played a role in this process. Through immunohistochemistry on human tissue microarrays, we showed that NLGN1 is expressed by prostatic and pancreatic cancer tissues in discrete stages and tumor districts. Next, we performed in vitro and in vivo assays, demonstrating that NLGN1 promotes cancer cell invasion and migration along nerves. Because of the established role of the neurotrophic factor glial cell line-derived neurotrophic factor (GDNF) in tumor-nerve interactions, we assessed a potential NLGN1-GDNF cooperation. We found that blocking GDNF activity with a specific antibody completely inhibited NLGN1-induced in vitro cancer cell invasion of nerves. Finally, we demonstrated that, in the presence of NLGN1, GDNF markedly activates cofilin, a cytoskeletal regulatory protein, altering filopodia dynamics. In conclusion, our data further prove the existence of a molecular and functional cross-talk between the nervous system and cancer cells. NLGN1 was shown here to function along one of the most represented neurotrophic factors in the nerve microenvironment, possibly opening new therapeutic avenues.

Modulation of Angiopoietin 2 release from endothelial cells and angiogenesis by the synaptic protein Neuroligin 2.

The synaptic protein Neuroligin 2, similarly to its isoform Neuroligin 1, is produced by endothelial cells, but its activity in the vascular context remains unknown. This study aimed at verifying the hypothesis that Neuroligin 2, in parallel with its extraneuronal involvement in pancreatic beta cells exocytosis, modulated cytokine release from endothelial cells and consequently angiogenesis. We used in vitro approaches to modulate Neuroligin 2 expression and Neuroligin 2 null mice to test our hypotheses. In vitro, upon VEGF stimulation, Neuroligin 2 silencing strongly reduces Angiopoietin 2 release in the medium and increases the endothelial cell retention of Weibel Palade Bodies, the specialized organelles that store Angiopoietin 2 and various other cytokines. On the contrary, Neuroligin 2 overexpression almost depletes cells of Weibel Palade Bodies, independent of VEGF. In vivo, both the retina and tumor xenografts grown in NLGN2- null mice display an immature vasculature, with lower pericyte coverage and lower Tie2 phosphorylation. At the molecular level NLGN2 colocalizes with its neuronal partner collibystin, a CDC42 guanine nucleotide exchange factor, which is also expressed by endothelial cells and in turn modulates Angiopoietin 2 release. Neuroligin 2, an inhibitory synaptic protein, modulates a peculiar aspect of vascular function and could represent a novel target of therapy in various fields, from tumor angiogenesis to vascular diseases.

Tumor progression: the neuronal input.

One of the challenges of cancer is its heterogeneity and rapid capacity to adapt. Notwithstanding significant progress in the last decades in genomics and precision medicine, new molecular targets and therapies appear highly necessary. One way to approach this complex problem is to consider cancer in the context of its cellular and molecular microenvironment, which includes nerves. The peripheral nerves, the topic of this review, modulate the biological behavior of the cancer cells and influence tumor progression, including the events related to the metastatic spread of the disease. This mechanism involves the release of neurotransmitters directly into the microenvironment and the activation of the corresponding membrane receptors. While this fact appears to complicate further the molecular landscape of cancer, the neurotransmitters are highly investigated molecules, and often are already targeted by well-developed drugs, a fact that can help finding new therapies at a fraction of the cost and time needed for new medicines (through the so-called drug repurposing). Moreover, the modulation of tumor progression by neurotransmitters can probably explain the long-recognized effects of psychological factors on the burden of cancer. We begin with an introduction on the tumor-nervous-connections and a description of the perineural invasion and neoneurogenesis, the two most important interaction patterns of cancer and nerves. Next, we discuss the most recent data that unequivocally demonstrate the necessity of the nervous system for tumor onset and growth. We introduce the molecular players of the tumor-nervous-connections by citing the role of three main families: neurotropic factors, axon guidance molecules, and neurotransmitters. Finally, we review the role the most important neurotransmitters in tumor biology and we conclude by analyzing the significance of the presented data for cancer therapy, with all the potential advantages and caveats.

Soluble Neuregulin1 is strongly up-regulated in the rat model of Charcot-Marie-Tooth 1A disease.

Neuregulin1 (NRG1) is a growth factor playing a pivotal role in peripheral nerve development through the activation of the transmembrane co-receptors ErbB2-ErbB3. Soluble NRG1 isoforms, mainly secreted by Schwann cells, are strongly and transiently up-regulated after acute peripheral nerve injury, thus suggesting that they play a crucial role also in the response to nerve damage. Here we show that in the rat experimental model of the peripheral demyelinating neuropathy Charcot-Marie-Tooth 1A (CMT1A) the expression of the different NRG1 isoforms (soluble, type α and ß, type a and b) is strongly up-regulated, as well as the expression of NRG1 co-receptors ErbB2-ErbB3, thus showing that CMT1A nerves have a gene expression pattern highly reminiscent of injured nerves. Because it has been shown that high concentrations of soluble NRG1 negatively affect myelination, we suggest that soluble NRG1 over-expression might play a negative role in the pathogenesis of CMT1A disease, and that a therapeutic approach, aimed to interfere with NRG1 activity, might be beneficial for CMT1A patients. Further studies will be necessary to test this hypothesis in animal models and to evaluate NRG1 expression in human patients. Impact statement Charcot-Marie-Tooth1A (CMT1A) is one of the most frequent inherited neurological diseases, characterized by chronic demyelination of peripheral nerves, for which effective therapies are not yet available. It has been recently proposed that the treatment with soluble Neuregulin1 (NRG1), a growth factor released by Schwann cells immediately after acute nerve injury, might be effective in CMT1A treatment. However, the expression of the different isoforms of endogenous NRG1 in CMT1A nerves has not been yet investigated. In this preliminary study, we demonstrate that different isoforms of soluble NRG1 are strongly over-expressed in CMT1A nerves, thus suggesting that a therapeutic approach based on NRG1 treatment should be carefully reconsidered. If soluble NRG1 is over-expressed also in human CMT1A nerves, a therapeutic approach aimed to inhibit (instead of stimulate) the signal transduction pathways driven by NRG1 might be fruitfully developed. Further studies will be necessary to test these hypotheses.

Efficacy of anti-adhesion gel of carboxymethylcellulose with polyethylene oxide on peripheral nerve: Experimental results on a mouse model.

INTRODUCTION: Perineural scar formation is responsible for pain and loss of function after surgical procedures. Neurolysis and application of anti-adhesion gels are required to restore a gliding surface. We tested a carboxymethylcellulose (CMC) and polyethylene oxide (PEO) gel on mouse sciatic nerve to describe its safety and efficacy. METHODS: Adult mice underwent a surgical procedure in which we burned the muscular bed of the sciatic nerve bilaterally (Burned group) and applied anti-adhesion gel to 1 of the nerves (Burned+gel group). After 3 weeks, we studied scar tissue by biomechanical and histological evaluation. RESULTS: Both histological and biomechanical analysis showed that the gel reduced perineural scarring. The difference between the Burned and Burned+gel groups was statistically significant. CONCLUSIONS: CMC-PEO gel can reduce perineural scar tissue. In histological section, scar tissue was present in both groups, but in the Burned+gel group a gliding surface was identified between scar and nerve.

Characterization of glial cell models and in vitro manipulation of the neuregulin1/ErbB system.

The neuregulin1/ErbB system plays an important role in Schwann cell behavior both in normal and pathological conditions. Upon investigation of the expression of the neuregulin1/ErbB system in vitro, we explored the possibility to manipulate the system in order to increase the migration of Schwann cells, that play a fundamental role in the peripheral nerve regeneration. Comparison of primary cells and stable cell lines shows that both primary olfactory bulb ensheathing cells and a corresponding cell line express ErbB1-ErbB2 and neuregulin1, and that both primary Schwann cells and a corresponding cell line express ErbB2-ErbB3, while only primary Schwann cells express neuregulin1. To interfere with the neuregulin1/ErbB system, the soluble extracellular domain of the neuregulin1 receptor ErbB4 (ecto-ErbB4) was expressed in vitro in the neuregulin1 expressing cell line, and an unexpected increase in cell motility was observed. In vitro experiments suggest that the back signaling mediated by the transmembrane neuregulin1 plays a role in the migratory activity induced by ecto-ErbB4. These results indicate that ecto-ErbB4 could be used in vivo as a tool to manipulate the neuregulin1/ErbB system.

A simple and reliable method to perform biomechanical evaluation of postoperative nerve adhesions.

BACKGROUND: Perineural fibrotic adhesions are among the major complications of peripheral nerve surgery. While different experimental models have been used for the pre-clinical testing of anti-adherential strategies, the methods used so far to induce scar tissue appear to be poorly standardized and reproducible. NEW METHOD: Thirty adult mice were used. Two methods were tested: the first one is based on burning the perineural muscular bed with a diathermocoagulator, while the second is based on direct scratching of the nerve surface with a cotton swab. After 3 weeks, the fibrotic reaction was assessed by measuring the peak pull out force of the nerve from muscular bed by means of a new tool specifically devised for biomechanical assessment of scar tissue formation. Moreover, histological analysis with specific collagen stain was also carried out. RESULTS: Both methods produced fibrotic reaction. Statistical analysis of biomechanical data showed a significant difference between burning and scratching group compared to the control sham operated group. No significant differences were detected between burning and scratching group. Histological analysis showed the presence of perineural scar tissue in both groups, though with a different distribution pattern. COMPARISON WITH OTHER METHODS: This protocol is easier to perform. The tool used for biomechanical evaluation is reliable and cheap. CONCLUSIONS: Both methods for perineural scar formation are effective and simple. They represent reproducible models for the study of the anti-adherential strategies. Yet, biomechanical testing with the device that we have developed proved to be a reliable and simple method for the quantitative assessment of the degree of perineural adhesion formation.

Ghrelin: a novel neuromuscular recovery promoting factor?

Promoting neuromuscular recovery after neural injury is a major clinical issue. While techniques for nerve reconstruction are continuously improving and most peripheral nerve lesions can be repaired today, recovery of the lost function is usually unsatisfactory. This evidence claims for innovative nonsurgical therapeutic strategies that can implement the outcome after neural repair. Although no pharmacological approach for improving posttraumatic neuromuscular recovery has still entered clinical practice, various molecules are explored in experimental models of neural repair. One of such molecules is the circulating peptide hormone ghrelin. This hormone has proved to have a positive effect on neural repair after central nervous system lesion, and very recently its effectiveness has also been demonstrated in preventing posttraumatic skeletal muscle atrophy. By contrast, no information is still available about its effectiveness on peripheral nerve regeneration although preliminary data from our laboratory suggest that this molecule can have an effect also in promoting axonal regeneration after nerve injury and repair. Should this be confirmed, ghrelin might represent an ideal candidate as a therapeutic agent for improving posttraumatic neuromuscular recovery because of its putative effects at all the various structural levels involved in this regeneration process, namely, the central nervous system, the peripheral nerve, and the target skeletal muscle.