RNA editing of CYFIP2 regulates actin related cellular migration and neuronal development in vitro

Cytoplasmic FMRP Interacting Protein 2 (CYFIP2) è una proteina originariamente identificata come componente del Wave Regulatory Complex e come interattore di Fragile-X Mental Retardation Protein. Il trascritto di CYFIP2 è sottoposto alla reazione di editing dell'RNA, un meccanismo post-trascrizionale catalizzato dagli enzimi ADAR, che porta l'Adenosina alla deaminazione a Inosina. In seguito al processo di traduzione, l'editing di CYFIP2 comporta una sostituzione K/E dell'amminoacido in posizione 320. Questo studio vuole indagare le potenziali implicazioni di questo processo nei fenomeni correlati alla dinamica dell'actina durante la migrazione cellulare, lo sviluppo degli assoni e la sinaptogenesi nelle cellule neurali. I nostri risultati mostrano che l’editing dell'RNA di CYFIP2 aumenta durante lo sviluppo neurale. È un processo attivo solo nel sistema nervoso centrale. Per ottenere maggiori informazioni sul ruolo dell'RNA di CYFIP2, abbiamo inizialmente eliminato il gene CYFIP2 in cellule di neuroblastoma e abbiamo sovraespresso le varianti di CYFIP2 non editate ed editate nelle linee KO. La sovra espressione di entrambe le varianti è in grado di ripristinare il fenotipo morfologico delle cellule analizzate. I nostri risultati mostrano differenze funzionali delle proteine CYFIP2 editate e non editate nella regolazione della migrazione cellulare. La variante editata presenta una maggiore capacità di migrare. Abbiamo inoltre investigato il ruolo delle varianti di CYFIP2 durante lo sviluppo neuronale. A tale scopo, abbiamo utilizzato un consolidato modello di differenziazione neuronale in cellule SH-SY5Y, basato sull'applicazione di acido retinoico (RA) e fattore di crescita neuronale (BDNF) per monitorare la conversione di cellule indifferenziato in cellule con morfologia simil-neuronale altamente polarizzata. I nostri risultati dimostrano chiaramente che le cellule KO per il gene CYFIP2 hanno perso la capacità di produrre una caratteristica morfologia simil-neuronale dopo il differenziamento, mentre la sovra espressione delle varianti di editing ne ripristina la capacità differenziativa. Tuttavia, non è stato possibile rilevare differenze tra le popolazioni di cellule K ed E, indicando come la proteina CYFIP2 sia importante per il differenziamento neuronale, ma il processo di editing potrebbe non avere un ruolo in questo processo. Per approfondire il ruolo delle varianti K e E in un sistema più fisiologico abbiamo deciso di utilizzare colture neuronali primarie di roditore. L'espressione di CYFIP2 endogeno è stata silenziata utilizzando specifici shRNA e le colture cellulari sono state trasdotte con vettori lentivirali esprimenti le varianti K o E di CYFIP2. Durante la maturazione neuronale sono stati analizzati parametri morfologici relativi allo sviluppo assonale delle cellule. Abbiamo riscontrato una differenza statisticamente significativa tra i neuroni che esprimono le varianti non editate ed editate nel numero di ramificazioni, nella lunghezza totale e nell'indice di complessità degli assoni analizzati. Abbiamo inoltre applicato il nostro modello basato sulle colture primarie ippocampali per cercare di capire se l’editing di CYFIP2 possa essere implicato nel processo di sinaptogenesi. I nostri risultati mostrano come la frequenza delle spine venga drasticamente ridotta nelle cellule in cui la proteina CYFIP2 è stata silenziata. La popolazione di cellule che esprimono in maggioranza la variante di CYFIP2 non editata (K) non portano ad un ripristino nella frequenza nelle spine che al contrario si osserva nelle cellule che si sviluppano esprimendo la variante editata di CYFIP2 (E). Nel loro insieme questi risultati mostrano un chiaro ruolo della proteina CYFIP2 nella regolazione dello sviluppo assonale durante le prime fasi dello sviluppo in vitro e durante il processo di sinaptogenesi. La reazione K/E di editing dell'RNA del trascritto risulta inoltre importante in entrambi i processi studiati.Cytoplasmic FMRP Interacting Protein 2 (CYFIP2) is a protein originally identified as a component of Wave Regulatory Complex (WRC) and Fragile-X Mental Retardation Protein (FMRP) interactor. CYFIP2 transcript undergoes RNA editing, a post-transcriptional mechanism catalysed by ADAR enzymes, that leads adenosine to inosine deamination. CYFIP2 editing results in a K/E substitution at amino acid 320. In this study, we aim at investigating the potential implication of this process related to actin dynamics during cell migration and axon development and synaptogenesis in neural cells. Understanding the role of CYFIP2 RNA editing substitution would be fundamental for further analysis in normal and pathological conditions. Our results show that CYFIP2 RNA editing reaction increases during neural development. It is active only in the central nervous system. Taken together these data indicate that a proper CYFIP2 editing regulation is important during neuronal development and function. We have generated neuroblastoma cell lines deleted of CYFIP2 gene. The absence of CYFIP2 clearly modifies SH-SY5Y cellular morphology, leading to acting dynamic dysregulation. We have overexpressed CYFIP2 unedited and edited variants in KO cell lines, using lentivirus transduction. The overexpression of both variants are able to revert the morphological phenotype. Our results show functional differences of the edited and unedited CYFIP2 proteins in regulating cellular migration after wound healing experiments. In particular, the edited variant (CYFIP2-E) presents a stronger capability to migrate and to invade the experimental wound, both in starvation conditions and under growth factors stimulation. These data indicate that the editing reaction might be involved in the acting dynamic underlining cellular migration and proliferation.Since CYFIP2 protein is mainly expressed in neurons and K/E RNA editing reaction is active only in the central nervous system, we investigate the role of CYFIP variants on neuronal development. For this purpose, we use a well-established model of neuronal differentiation based on the application on SH-SY5Y cells of a two-step retinoic acid (RA) and brain-derived neurotrophic factor (BDNF) treatment to monitor the conversion of undifferentiated SH-SY5Y into neuron-like cells with distinctly polarized axon-dendritic morphology. Our results clearly demonstrate that KO cells have lost their ability to produce a characteristic neurite-like morphology after differentiation. However, no differences could be detected between K and E cell populations, indicating that CYFIP2 is important for neuronal differentiation but RNA editing isoform might have a similar function in this process for SH-SY5Y.Since the effect of K/E variants resulted irrelevant in neural maturation process using a previously described model, we took advantage of primary neuronal culture to try to understand the effect of CYFIP2 variants on neuronal maturation and spine dynamics. The expression of endogenous CYFIP2 has been down-regulated by specific shRNA and the cell culture has been transduced with lentiviral vectors expressing CYFIP2 K/E variants. During neuronal maturation a morphological parameters concerning axon development has been analysed. We found a statistically significant difference between neurons carrying K and E variants in the number of axon branches, total axon length and axon complexity index. Taken together these results suggest a clear role of CYFIP2 K/E RNA editing process in regulating the spreading of neuron axon during first stages of in-vitro development.These findings suggest that the K/E editing variations have a functional role that needs further investigation. Additionally, an in-vivo study can be useful to understand the role of CYFIP2 editing variants during neural development and function

Differential Enzymatic Activity of Rat ADAR2 Splicing Variants Is Due to Altered Capability to Interact with RNA in the Deaminase Domain

In mammals, adenosine (A) to inosine (I) RNA editing is performed by adenosine deaminases acting on RNA (ADAR), ADAR1 and ADAR2 enzymes, encoded by mRNAs that might undergo splicing process. In rat, two splicing events produce several isoforms of ADAR2, called ADAR2a, ADAR2b, ADAR2e, and ADAR2f, but only ADAR2a and ADAR2b are translated into an active protein. In particular, they differ for ten amino acids located in the catalytic domain of ADAR2b. Here, we focused on these two isoforms, analyzing the splicing pattern and their different function during rat neuronal maturation. We found an increase of editing levels in cortical neurons overexpressing ADAR2a compared to those overexpressing ADAR2b. These results indicate ADAR2a isoform as the most active one, as reported for the homologous human short variant. Furthermore, we showed that the differential editing activity is not due to a different dimerization of the two isoforms; it seems to be linked to the ten amino acids loop of ADAR2b that might interfere with RNA binding, occupying the space volume in which the RNA should be present in case of binding. These data might shed light on the complexity of ADAR2 regulations

Restoration by ketamine of stress-induced maladaptive changes in synaptic function and brain architecture

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Transcriptional Profiling of Rat Prefrontal Cortex after Acute Inescapable Footshock Stress

Stress is a primary risk factor for psychiatric disorders such as Major Depressive Disorder (MDD) and Post Traumatic Stress Disorder (PTSD). The response to stress involves the regulation of transcriptional programs, which is supposed to play a role in coping with stress. To evaluate transcriptional processes implemented after exposure to unavoidable traumatic stress, we applied microarray expression analysis to the PFC of rats exposed to acute footshock (FS) stress that were sacrificed immediately after the 40 min session or 2 h or 24 h after. While no substantial changes were observed at the single gene level immediately after the stress session, gene set enrichment analysis showed alterations in neuronal pathways associated with glia development, glia-neuron networking, and synaptic function. Furthermore, we found alterations in the expression of gene sets regulated by specific transcription factors that could represent master regulators of the acute stress response. Of note, these pathways and transcriptional programs are activated during the early stress response (immediately after FS) and are already turned off after 2 h-while at 24 h, the transcriptional profile is largely unaffected. Overall, our analysis provided a transcriptional landscape of the early changes triggered by acute unavoidable FS stress in the PFC of rats, suggesting that the transcriptional wave is fast and mild, but probably enough to activate a cellular response to acute stress

Alpha-synuclein/synapsin III pathological interplay boosts the motor response to methylphenidate

: Loss of dopaminergic nigrostriatal neurons and fibrillary α-synuclein (α-syn) aggregation in Lewy bodies (LB) characterize Parkinson's disease (PD). We recently found that Synapsin III (Syn III), a phosphoprotein regulating dopamine (DA) release with α-syn, is another key component of LB fibrils in the brain of PD patients and acts as a crucial mediator of α-syn aggregation and toxicity. Methylphenidate (MPH), a monoamine reuptake inhibitor (MRI) efficiently counteracting freezing of gait in advanced PD patients, can bind α-syn and controls α-syn-mediated DA overflow and presynaptic compartmentalization. Interestingly, MPH results also efficient for the treatment of attention deficits and hyperactivity disorder (ADHD), a neurodevelopmental psychiatric syndrome associated with Syn III and α-syn polymorphisms and constituting a risk factor for the development of LB disorders. Here, we studied α-syn/Syn III co-deposition and longitudinal changes of α-syn, Syn III and DA transporter (DAT) striatal levels in nigrostriatal neurons of a PD model, the human C-terminally truncated (1-120) α-syn transgenic (SYN120 tg) mouse, in comparison with C57BL/6J wild type (wt) and C57BL/6JOlaHsd α-syn null littermates. Then, we analyzed the locomotor response of these animals to an acute administration of MPH (d-threo) and other MRIs: cocaine, that we previously found to stimulate Syn III-reliant DA release in the absence of α-syn, or the selective DAT blocker GBR-12935, along aging. Finally, we assessed whether these drugs modulate α-syn/Syn III interaction by fluorescence resonance energy transfer (FRET) and performed in silico studies engendering a heuristic model of the α-syn conformations stabilized upon MPH binding. We found that only MPH was able to over-stimulate a Syn III-dependent/DAT-independent locomotor activity in the aged SYN120 tg mice showing α-syn/Syn III co-aggregates. MPH enhanced full length (fl) α-syn/Syn III and even more (1-120) α-syn/Syn III interaction in cells exhibiting α-syn/Syn III inclusions. Moreover, in silico studies confirmed that MPH may reduce α-syn fibrillation by stabilizing a protein conformation with increased lipid binding predisposition. Our observations indicate that the motor-stimulating effect of MPH can be positively fostered in the presence of α-syn/Syn III co-aggregation. This evidence holds significant implications for PD and ADHD therapeutic management

Impaired activation of plasmacytoid dendritic cells via toll-like receptor 7/9 and STING is mediated by melanoma-derived immunosuppressive cytokines and metabolic drift

IntroductionPlasmacytoid dendritic cells (pDCs) infiltrate a large set of human cancers. Interferon alpha (IFN-α) produced by pDCs induces growth arrest and apoptosis in tumor cells and modulates innate and adaptive immune cells involved in anti-cancer immunity. Moreover, effector molecules exert tumor cell killing. However, the activation state and clinical relevance of pDCs infiltration in cancer is still largely controversial. In Primary Cutaneous Melanoma (PCM), pDCs density decreases over disease progression and collapses in metastatic melanoma (MM). Moreover, the residual circulating pDC compartment is defective in IFN-α production.MethodsThe activation of tumor-associated pDCs was evaluated by in silico and microscopic analysis. The expression of human myxovirus resistant protein 1 (MxA), as surrogate of IFN-α production, and proximity ligation assay (PLA) to test dsDNA-cGAS activation were performed on human melanoma biopsies. Moreover, IFN-α and CXCL10 production by in vitro stimulated (i.e. with R848, CpG-A, ADU-S100) pDCs exposed to melanoma cell lines supernatants (SN-mel) was tested by intracellular flow cytometry and ELISA. We also performed a bulk RNA-sequencing on SN-mel-exposed pDCs, resting or stimulated with R848. Glycolytic rate assay was performed on SN-mel-exposed pDCs using the Seahorse XFe24 Extracellular Flux Analyzer.ResultsBased on a set of microscopic, functional and in silico analyses, we demonstrated that the melanoma milieu directly impairs IFN-α and CXCL10 production by pDCs via TLR-7/9 and cGAS-STING signaling pathways. Melanoma-derived immunosuppressive cytokines and a metabolic drift represent relevant mechanisms enforcing pDC-mediated melanoma escape.DiscussionThese findings propose a new window of intervention for novel immunotherapy approaches to amplify the antitumor innate immune response in cutaneous melanoma (CM)

Sicilia—silicon carbide detectors for intense luminosity investigations and applications

Silicon carbide (SiC) is a compound semiconductor, which is considered as a possible alternative to silicon for particles and photons detection. Its characteristics make it very promising for the next generation of nuclear and particle physics experiments at high beam luminosity. Silicon Carbide detectors for Intense Luminosity Investigations and Applications (SiCILIA) is a project starting as a collaboration between the Italian National Institute of Nuclear Physics (INFN) and IMM-CNR, aiming at the realization of innovative detection systems based on SiC. In this paper, we discuss the main features of silicon carbide as a material and its potential application in the field of particles and photons detectors, the project structure and the strategies used for the prototype realization, and the first results concerning prototype production and their performance

Defining Kawasaki disease and pediatric inflammatory multisystem syndrome-temporally associated to SARS-CoV-2 infection during SARS-CoV-2 epidemic in Italy: results from a national, multicenter survey

Background: There is mounting evidence on the existence of a Pediatric Inflammatory Multisystem Syndrome-temporally associated to SARS-CoV-2 infection (PIMS-TS), sharing similarities with Kawasaki Disease (KD). The main outcome of the study were to better characterize the clinical features and the treatment response of PIMS-TS and to explore its relationship with KD determining whether KD and PIMS are two distinct entities. Methods: The Rheumatology Study Group of the Italian Pediatric Society launched a survey to enroll patients diagnosed with KD (Kawasaki Disease Group - KDG) or KD-like (Kawacovid Group - KCG) disease between February 1st 2020, and May 31st 2020. Demographic, clinical, laboratory data, treatment information, and patients' outcome were collected in an online anonymized database (RedCAP®). Relationship between clinical presentation and SARS-CoV-2 infection was also taken into account. Moreover, clinical characteristics of KDG during SARS-CoV-2 epidemic (KDG-CoV2) were compared to Kawasaki Disease patients (KDG-Historical) seen in three different Italian tertiary pediatric hospitals (Institute for Maternal and Child Health, IRCCS "Burlo Garofolo", Trieste; AOU Meyer, Florence; IRCCS Istituto Giannina Gaslini, Genoa) from January 1st 2000 to December 31st 2019. Chi square test or exact Fisher test and non-parametric Wilcoxon Mann-Whitney test were used to study differences between two groups. Results: One-hundred-forty-nine cases were enrolled, (96 KDG and 53 KCG). KCG children were significantly older and presented more frequently from gastrointestinal and respiratory involvement. Cardiac involvement was more common in KCG, with 60,4% of patients with myocarditis. 37,8% of patients among KCG presented hypotension/non-cardiogenic shock. Coronary artery abnormalities (CAA) were more common in the KDG. The risk of ICU admission were higher in KCG. Lymphopenia, higher CRP levels, elevated ferritin and troponin-T characterized KCG. KDG received more frequently immunoglobulins (IVIG) and acetylsalicylic acid (ASA) (81,3% vs 66%; p = 0.04 and 71,9% vs 43,4%; p = 0.001 respectively) as KCG more often received glucocorticoids (56,6% vs 14,6%; p < 0.0001). SARS-CoV-2 assay more often resulted positive in KCG than in KDG (75,5% vs 20%; p < 0.0001). Short-term follow data showed minor complications. Comparing KDG with a KD-Historical Italian cohort (598 patients), no statistical difference was found in terms of clinical manifestations and laboratory data. Conclusion: Our study suggests that SARS-CoV-2 infection might determine two distinct inflammatory diseases in children: KD and PIMS-TS. Older age at onset and clinical peculiarities like the occurrence of myocarditis characterize this multi-inflammatory syndrome. Our patients had an optimal response to treatments and a good outcome, with few complications and no deaths

Search for dark matter produced in association with bottom or top quarks in √s = 13 TeV pp collisions with the ATLAS detector

A search for weakly interacting massive particle dark matter produced in association with bottom or top quarks is presented. Final states containing third-generation quarks and miss- ing transverse momentum are considered. The analysis uses 36.1 fb−1 of proton–proton collision data recorded by the ATLAS experiment at √s = 13 TeV in 2015 and 2016. No significant excess of events above the estimated backgrounds is observed. The results are in- terpreted in the framework of simplified models of spin-0 dark-matter mediators. For colour- neutral spin-0 mediators produced in association with top quarks and decaying into a pair of dark-matter particles, mediator masses below 50 GeV are excluded assuming a dark-matter candidate mass of 1 GeV and unitary couplings. For scalar and pseudoscalar mediators produced in association with bottom quarks, the search sets limits on the production cross- section of 300 times the predicted rate for mediators with masses between 10 and 50 GeV and assuming a dark-matter mass of 1 GeV and unitary coupling. Constraints on colour- charged scalar simplified models are also presented. Assuming a dark-matter particle mass of 35 GeV, mediator particles with mass below 1.1 TeV are excluded for couplings yielding a dark-matter relic density consistent with measurements

Mortality and pulmonary complications in patients undergoing surgery with perioperative SARS-CoV-2 infection: an international cohort study

Background: The impact of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on postoperative recovery needs to be understood to inform clinical decision making during and after the COVID-19 pandemic. This study reports 30-day mortality and pulmonary complication rates in patients with perioperative SARS-CoV-2 infection. Methods: This international, multicentre, cohort study at 235 hospitals in 24 countries included all patients undergoing surgery who had SARS-CoV-2 infection confirmed within 7 days before or 30 days after surgery. The primary outcome measure was 30-day postoperative mortality and was assessed in all enrolled patients. The main secondary outcome measure was pulmonary complications, defined as pneumonia, acute respiratory distress syndrome, or unexpected postoperative ventilation. Findings: This analysis includes 1128 patients who had surgery between Jan 1 and March 31, 2020, of whom 835 (74·0%) had emergency surgery and 280 (24·8%) had elective surgery. SARS-CoV-2 infection was confirmed preoperatively in 294 (26·1%) patients. 30-day mortality was 23·8% (268 of 1128). Pulmonary complications occurred in 577 (51·2%) of 1128 patients; 30-day mortality in these patients was 38·0% (219 of 577), accounting for 81·7% (219 of 268) of all deaths. In adjusted analyses, 30-day mortality was associated with male sex (odds ratio 1·75 [95% CI 1·28–2·40], p\textless0·0001), age 70 years or older versus younger than 70 years (2·30 [1·65–3·22], p\textless0·0001), American Society of Anesthesiologists grades 3–5 versus grades 1–2 (2·35 [1·57–3·53], p\textless0·0001), malignant versus benign or obstetric diagnosis (1·55 [1·01–2·39], p=0·046), emergency versus elective surgery (1·67 [1·06–2·63], p=0·026), and major versus minor surgery (1·52 [1·01–2·31], p=0·047). Interpretation: Postoperative pulmonary complications occur in half of patients with perioperative SARS-CoV-2 infection and are associated with high mortality. Thresholds for surgery during the COVID-19 pandemic should be higher than during normal practice, particularly in men aged 70 years and older. Consideration should be given for postponing non-urgent procedures and promoting non-operative treatment to delay or avoid the need for surgery. Funding: National Institute for Health Research (NIHR), Association of Coloproctology of Great Britain and Ireland, Bowel and Cancer Research, Bowel Disease Research Foundation, Association of Upper Gastrointestinal Surgeons, British Association of Surgical Oncology, British Gynaecological Cancer Society, European Society of Coloproctology, NIHR Academy, Sarcoma UK, Vascular Society for Great Britain and Ireland, and Yorkshire Cancer Research