A preliminary study on the role of Piezo1 channels in myokine release from cultured mouse myotubes

It has long been known that regular physical exercise induces short and long term benefits reducing the risk of cardiovascular disease, diabetes, osteoporosis, cancer and improves sleep quality, cognitive level, mobility, autonomy in enderly. More recent is the evidence on the endocrine role of the contracting skeletal muscle. Exercise triggers the release of miokines, which act in autocrine, paracrine and endocrine ways controlling the activity of muscles but also of other tissues and organs such as adipose tissue, liver, pancreas, bones, and brain. The mechanism of release is still unclear. Neuromuscular electrical stimulation reproduces the beneficial effects of physical activity producing physiological metabolic, cardiovascular, aerobic responses consistent with those induced by exercise. In vitro, Electrical Pulse Stimulations (EPS) of muscle cells elicit cell contraction and mimic miokine release in the external medium. Here we show that, in cultured mouse myotubes, EPS induce contractile activity and the release of the myokine IL-6. Gadolinium highly reduces EPS-induced IL-6 release, suggesting the involvement of mechanical activated ion channels. The chemical activation of mechanosensitive Piezo1 channels with the specific agonist Yoda1 stimulates IL-6 release similarly to EPS, suggesting the involvement of Piezo1 channels in the control of the myokine release. The expression of Piezo1 protein in myotubes was confirmed by the Western blot analysis. To the best of our knowledge, this is the first evidence of a Piezo1-mediated effect in myokine release and suggests a potential translational use of specific Piezo1 agonists for innovative therapeutic treatments reproducing/enhancing the benefits of exercise mediated by myokines

Asbestos Fibers Enhance the TMEM16A Channel Activity in Xenopus Oocytes

Background: The interaction of asbestos fibers with target cell membranes is still poorly investigated. Here, we detected and characterized an enhancement of chloride conductance in Xenopus oocyte cell membranes induced by exposure to crocidolite (Croc) asbestos fibers. Methods: A two-microelectrode voltage clamp technique was used to test the effect of Croc fiber suspensions on outward chloride currents evoked by step membrane depolarization. Calcium imaging experiments were also performed to investigate the variation of ‘resting’ oocyte [Ca2+]i following asbestos exposure. Results: The increase in chloride current after asbestos treatment, was sensitive to [Ca2+]e, and to specific blockers of TMEM16A Ca2+-activated chloride channels, MONNA and Ani9. Furthermore, asbestos treatment elevated the ‘resting’ [Ca2+]i likelihood by increasing the cell membrane permeability to Ca2 in favor of a tonic activation of TMEME16A channels. Western blot analysis confirmed that TMEME16A protein was endogenously present in the oocyte cell membrane and absorbed by Croc. Conclusion: the TMEM16A channels endogenously expressed by Xenopus oocytes are targets for asbestos fibers and represent a powerful tool for asbestos–membrane interaction studies. Interestingly, TMEM16A channels are highly expressed in many types of tumors, including some asbestos-related cancers, suggesting them, for the first time, as a possible early target of crocidolite-mediated tumorigenic effects on target cell membranes

Declusterization of GABAA receptors affects the kinetic properties of GABAergic currents in cultured hippocampal neurons.

Speed and reliability of synaptic transmission are essential for information coding in neuronal networks and require the presence of clustered neurotransmitter receptors at the plasma membrane in precise apposition to presynaptic terminals. Receptor clusterization is the result of highly regulated processes involving functional and structural proteins. Among the structural elements, microtubules are known to play a crucial role in anchoring of gamma-aminobutyric acid, type A (GABA(A)) receptors. Here we show that microtubule depolymerization with nocodazole induces the declusterization of GABA(A) receptors and modifies the kinetic properties of GABAergic currents in cultured hippocampal neurons. In particular, this drug, applied either in the bath or via the patch pipette, induced the acceleration of the onset kinetics of miniature inhibitory postsynaptic currents (mIPSCs) without significantly affecting their frequency, thus suggesting a main postsynaptic site of action. After nocodazole treatment, current responses to ultrafast applications of GABA exhibited a faster rise time and an accelerated onset of desensitization. A quantitative analysis of GABA-evoked currents and model simulations suggest that declusterization affects the gating properties of GABA(A) receptors. In particular, a faster entry into the desensitized state of declustered GABA(A) receptors may account for the changes in the kinetic properties of mIPSCs after nocodazole treatment. Hence it appears that the clustered condition of GABA(A) receptors contributes in shaping GABAergic currents

Tuning GABAergic Inhibition: Gephyrin Molecular Organization and Functions

To be highly reliable, synaptic transmission needs postsynaptic receptors (Rs) in precise apposition to the pre - synaptic release sites. At inhibitory synapses, the postsynaptic protein gephyrin self -assembles to form a scaffold that anchors glycine and GABA A Rs to the cytoskeleton, thus ensuring the accurate accumulation of postsynaptic receptors at the right place. This protein undergoes several post -translational modifications which control protein-protein interac- tion and downstream signaling pathways. In addition, through the constant exchange of scaffolding elements and recep- tors in and out of synapses, gephyrin dynamically regulates synaptic strength and plasticity.The aim of the present review is to highlight recent findings on the functional role of gephyrin at GABAergic inhibitory synapses. We will discuss different approaches used to interfere with gephyrin in order to unveil its function. In addition, we will focus on the impact of gephyrin structure and distribution at the nanoscale level on the functional properties of inhibitory synapses as well as the implications of this scaffold protein in synaptic plasticity processes. Finally, we will emphasize how gephyrin genetic mutations or alterations in protein expression levels are implicated in several neuropathological disorders, including aut- ism spectrum disorders, schizophrenia, temporal lobe epilepsy and Alzheimer's disease, all associated with severe def- icits of GABAergic signaling. This article is part of a Special Issue entitled: Honoring Ricardo Miledi - outstanding neuroscientist of XX-XXI centuries. (c) 2019 The Authors. Published by Elsevier Ltd on behalf of IBRO. This is an open access article under the CC BY -NC -ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)

Pin1 Modulates the Synaptic Content of NMDA Receptors via Prolyl-Isomerization of PSD-95

Phosphorylation of serine/threonine residues preceding a proline regulates the fate of its targets through postphosphorylation conformational changes catalyzed by the peptidyl-prolyl cis-/trans isomerase Pin1. By flipping the substrate between two different functional conformations, this enzyme exerts a fine-tuning of phosphorylation signals. Pin1 has been detected in dendritic spines and shafts where it regulates protein synthesis required to sustain the late phase of long-term potentiation (LTP). Here, we demonstrate that Pin1 residing in postsynaptic structures can interact with postsynaptic density protein-95 (PSD-95), a key scaffold protein that anchors NMDA receptors (NMDARs) in PSD via GluN2-type receptor subunits. Pin1 recruitment by PSD-95 occurs at specific serine-threonine/proline consensus motifs localized in the linker region connecting PDZ2 to PDZ3 domains. Upon binding, Pin1 triggers structural changes in PSD-95, thus negatively affecting its ability to interact with NMDARs. In electrophysiological experiments, larger NMDA-mediated synaptic currents, evoked in CA1 principal cells by Schaffer collateral stimulation, were detected in hippocampal slices obtained from Pin1(-/-) mice compared with controls. Similar results were obtained in cultured hippocampal cells expressing a PSD-95 mutant unable to undergo prolyl-isomerization, thus indicating that the action of Pin1 on PSD-95 is critical for this effect. In addition, an enhancement in spine density and size was detected in CA1 principal cells of Pin1(-/-) or in Thy-1GFP mice treated with the pharmacological inhibitor of Pin1 catalytic activity PiB.Our data indicate that Pin1 controls synaptic content of NMDARs via PSD-95 prolyl-isomerization and the expression of dendritic spines, both required for LTP maintenance

Inverse psoriasis in patient treated with atezolizumab

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Gephyrin Selective Intrabodies as a New Strategy for Studying Inhibitory Receptor Clustering

The microtubule-binding protein gephyrin is known to play a pivotal role in targeting and clustering postsynaptic inhibitory receptors. Here, the Intracellular Antibodies Capture Technology (IATC) was used to select two single-chain antibody fragments or intrabodies, which, fused to nuclear localization signals (NLS), were able to efficiently and selectively remove gephyrin from glycine receptor (GlyR) clusters. Co-transfection of NLS-tagged individual intrabodies with gephyrin-enhanced green fluorescent protein (EGFP) in HEK 293 cells revealed a partial relocalization of gephyrin aggregates onto the nucleus or in the perinuclear area. When expressed in cultured neurons, these intrabodies caused a significant reduction in the number of immunoreactive GlyR clusters, which was associated with a decrease in the peak amplitude of glycine-evoked whole cell currents as assessed with electrophysiological experiments. Hampering protein function at a posttranslational level may represent an attractive alternative for interfering with gephyrin function in a more spatially localized manner

Variant Enrichment Analysis to Explore Pathways Disruption in a Necropsy Series of Asbestos-Exposed Shipyard Workers

The variant enrichment analysis (VEA), a recently developed bioinformatic workflow, has been shown to be a valuable tool for whole-exome sequencing data analysis, allowing finding differences between the number of genetic variants in a given pathway compared to a reference dataset. In a previous study, using VEA, we identified different pathway signatures associated with the development of pulmonary toxicities in mesothelioma patients treated with radical hemithoracic radiation therapy. Here, we used VEA to discover novel pathways altered in individuals exposed to asbestos who developed or not asbestos-related diseases (lung cancer or mesothelioma). A population-based autopsy study was designed in which asbestos exposure was evaluated and quantitated by investigating objective signs of exposure. We selected patients with similar exposure to asbestos. Formalin-fixed paraffin-embedded (FFPE) tissues were used as a source of DNA and whole-exome sequencing analysis was performed, running VEA to identify potentially disrupted pathways in individuals who developed thoracic cancers induced by asbestos exposure. By using VEA analysis, we confirmed the involvement of pathways considered as the main culprits for asbestos-induced carcinogenesis: oxidative stress and chromosome instability. Furthermore, we identified protective genetic assets preserving genome stability and susceptibility assets predisposing to a worst outcome.This research was funded by grants from the Italian League for the Fight Against Cancer (LILT), ASSOCIAZIONE ISONTINA LILT (Bando di Ricerca sanitaria 2017-programma 5 per mille anno 2015) and Municipality of Monfalcone (Gorizia); Regione Autonoma Friuli-Venezia Giulia, Assessorato alla Salute e Protezione Sociale, LR 22/2001 (decree 1124/SPS, 09/20/2016, No. 1299); Institute for Maternal and Child Health IRCCS “Burlo Garofolo/Italian Ministry of Health” (BioHub 03/20); Interreg Italia-Slovenia, ISE-EMH 07/2019; and by Conselho Nacional de Desenvolvimento Científico e Tencológico (CNPq) from Brazil (311415/2020-2)

Complement protein C1q stimulates hyaluronic acid degradation via gC1qR/HABP1/p32 in malignant pleural mesothelioma

Complement component C1q can act as a pro-tumorigenic factor in the tumor microenvironment (TME). The TME in malignant pleural mesothelioma (MPM) is rich in C1q and hyaluronic acid (HA), whose interaction enhances adhesion, migration and proliferation of malignant cells. HA-bound C1q is also capable of modulating HA synthesis. Thus, we investigated whether HA-C1q interaction would affect HA degradation, analyzing the main degradation enzymes, hyaluronidase (HYAL)1 and HYAL2, and a C1q receptor candidate. We first proceeded with the characterization of HYALs in MPM cells, especially HYAL2, since bioinformatics survival analysis revealed that higher HYAL2 mRNA levels have an unfavorable prognostic index in MPM patients. Interestingly, Real-Time quantitative PCR, flow cytometry and Western blot highlighted an upregulation of HYAL2 after seeding of primary MPM cells onto HA-bound C1q. In an attempt to unveil the receptors potentially involved in HA-C1q signaling, a striking co-localization between HYAL2 and globular C1q receptor/HABP1/p32 (gC1qR) was found by immunofluorescence, surface biotinylation and proximity ligation assays. RNA interference experiments revealed a potentially regulatory function exerted by gC1qR on HYAL2 expression, since C1QBP (gene for gC1qR) silencing unexpectedly caused HYAL2 downregulation. In addition, the functional blockage of gC1qR by a specific antibody hindered HA-C1q signaling and prevented HYAL2 upregulation. Thus, C1q-HA interplay is responsible for enhanced HYAL2 expression, suggesting an increased rate of HA catabolism and the release of pro-inflammatory and pro-tumorigenic HA fragments in the MPM TME. Our data support the notion of an overall tumor-promoting property of C1q. Moreover, the overlapping localization and physical interaction between HYAL2 and gC1qR suggests a potential regulatory effect of gC1qR within a putative HA-C1q macromolecular complex

Is the complement protein C1q a pro- or anti-tumorigenic factor? Bioinformatics analysis involving human carcinomas

C1q is the first subcomponent of the classical pathway of the complement system and belongs to the C1q/Tumor Necrosis Factor superfamily. C1q can perform a diverse range of immune and non-immune functions in a complement-dependent as well as -independent manner. Being a pattern recognition molecule of the innate immunity, C1q can recognize a number of self, non-self and altered-self ligands and bring about effector mechanisms designed to clear pathogens via opsonisation and inflammatory response. C1q is locally synthesized by macrophages and dendritic cells, and thus, can get involved in a range of biological processes, such as angiogenesis and tissue remodeling, immune modulation, and immunologic tolerance. The notion of C1q involvement in the pathogenesis of cancer is still evolving. C1q appears to have a dual role in cancer: tumor promoting as well as tumor-protective, depending on the context of the disease. In the current study, we performed a bioinformatics analysis to investigate whether C1q can serve as a potential prognostic marker for human carcinoma. We used the Oncomine database and the survival analysis platforms Kaplan-Meier plotter. Our results showed that high levels of C1q have a favorable prognostic index in basal-like breast cancer for disease-free survival, and in HER2-positive breast cancer for overall survival, while it showed a pro-tumorigenic role of C1q in lung adenocarcinoma, and in clear cell renal cell carcinoma. This in silico study, if validated via a retrospective study, can be a step forward in establishing C1q as a new tool as a prognostic biomarker for various carcinoma.Institute for Maternal and Child Health, IRCCS Burlo Garofolo, Trieste, Ital