ALS-Associated TDP-43 Dysfunction Compromises UPF1-Dependent mRNA Metabolism Pathways Including Alternative Polyadenylation and 3'UTR Length.

UPF1-mediated decay entails several mRNA surveillance pathways that play a crucial role in cellular homeostasis. However, the precise role of UPF1 in postmitotic neurons remains unresolved, as does its activity in amyotrophic lateral sclerosis (ALS), a devastating neurodegenerative disease characterized by TDP-43 pathology and disrupted mRNA metabolism. Here, we used human iPSC-derived spinal motor neurons (MNs) to identify mRNAs subject to UPF1 degradation by integrating RNA-seq before and after UPF1 knockdown with RIP-seq to identify RNAs that co-immunoprecipitate with the active form of phosphorylated UPF1. We define a stringent set of bona fide UPF1 targets in MNs that are functionally enriched for autophagy and structurally enriched for GC-rich and long 3' UTRs but not for premature termination codon (PTC)-containing transcripts. TDP-43 depletion in iPSC-derived MNs reduces UPF1 phosphorylation and consequently post-transcriptional upregulation of UPF1 targets, suggesting that TDP-43 dysfunction compromises UPF1-mediated mRNA surveillance. Intriguingly, our datasets reveal that UPF1 and TDP-43 regulate alternative polyadenylation and 3'UTR length of mRNAs associated with synaptic and axonal function, a process that we find to be compromised in ALS models in vitro and ALS patient tissue. Our study provides a comprehensive description of UPF1-mediated mRNA decay activity in neurons, reveals overlapping roles between UPF1 and TDP-43 in regulating 3'UTR length, and offers novel insight into the intricate interplay between RNA metabolism and neurodegeneration in ALS.

Early clonal extinction in glioblastoma progression revealed by genetic barcoding.

Glioblastoma progression in its early stages remains poorly understood. Here, we transfer PDGFB and genetic barcodes in mouse brain to initiate gliomagenesis and enable direct tracing of glioblastoma evolution from its earliest possible stage. Unexpectedly, we observe a high incidence of clonal extinction events and progressive divergence in clonal sizes, even after the acquisition of malignant phenotype. Computational modeling suggests these dynamics result from clonal-based cell-cell competition. Through bulk and single-cell transcriptome analyses, coupled with lineage tracing, we reveal that Myc transcriptional targets have the strongest correlation with clonal size imbalances. Moreover, we show that the downregulation of Myc expression is sufficient to drive competitive dynamics in intracranially transplanted gliomas. Our findings provide insights into glioblastoma evolution that are inaccessible using conventional retrospective approaches, highlighting the potential of combining clonal tracing and transcriptomic analyses in this field.

Loss of function of the ALS-associated NEK1 kinase disrupts microtubule homeostasis and nuclear import.

Loss-of-function variants in NIMA-related kinase 1 (NEK1) constitute a major genetic cause of amyotrophic lateral sclerosis (ALS), accounting for 2 to 3% of all cases. However, how NEK1 mutations cause motor neuron (MN) dysfunction is unknown. Using mass spectrometry analyses for NEK1 interactors and NEK1-dependent expression changes, we find functional enrichment for proteins involved in the microtubule cytoskeleton and nucleocytoplasmic transport. We show that α-tubulin and importin-ß1, two key proteins involved in these processes, are phosphorylated by NEK1 in vitro. NEK1 is essential for motor control and survival in Drosophila models in vivo, while using several induced pluripotent stem cell (iPSC)-MN models, including NEK1 knockdown, kinase inhibition, and a patient mutation, we find evidence for disruptions in microtubule homeostasis and nuclear import. Notably, stabilizing microtubules with two distinct classes of drugs restored NEK1-dependent deficits in both pathways. The capacity of NEK1 to modulate these processes that are critically involved in ALS pathophysiology renders this kinase a formidable therapeutic candidate.

Specificity, Safety, Efficacy of EGFRvIII-Retargeted Oncolytic HSV for Xenotransplanted Human Glioblastoma.

Glioblastoma is a lethal primary brain tumor lacking effective therapy. The secluded onset site, combined with the infiltrative properties of this tumor, require novel targeted therapies. In this scenario, the use of oncolytic viruses retargeted to glioblastoma cells and able to spread across the tumor cells represent an intriguing treatment strategy. Here, we tested the specificity, safety and efficacy of R-613, the first oncolytic HSV fully retargeted to EGFRvIII, a variant of the epidermal growth factor receptor carrying a mutation typically found in glioblastoma. An early treatment with R-613 on orthotopically transplanted EGFRvIII-expressing human glioblastoma significantly increased the median survival time of mice. In this setting, the growth of human glioblastoma xenotransplants was monitored by a secreted luciferase reporter and showed that R-613 is able to substantially delay the development of the tumor masses. When administered as late treatment to a well-established glioblastomas, R-613 appeared to be less effective. Notably the uninfected tumor cells derived from the explanted tumor masses were still susceptible to R-613 infection ex vivo, thus suggesting that multiple treatments could enhance R-613 therapeutic efficacy, making R-613 a promising oncolytic HSV candidate for glioblastoma treatment.

M2 Muscarinic Receptor Activation Impairs Mitotic Progression and Bipolar Mitotic Spindle Formation in Human Glioblastoma Cell Lines.

BACKGROUND: Glioblastoma multiforme (GBM) is characterized by several genetic abnormalities, leading to cell cycle deregulation and abnormal mitosis caused by a defective checkpoint. We previously demonstrated that arecaidine propargyl ester (APE), an orthosteric agonist of M2 muscarinic acetylcholine receptors (mAChRs), arrests the cell cycle of glioblastoma (GB) cells, reducing their survival. The aim of this work was to better characterize the molecular mechanisms responsible for this cell cycle arrest. METHODS: The arrest of cell proliferation was evaluated by flow cytometry analysis. Using immunocytochemistry and time-lapse analysis, the percentage of abnormal mitosis and aberrant mitotic spindles were assessed in both cell lines. Western blot analysis was used to evaluate the modulation of Sirtuin2 and acetylated tubulin-factors involved in the control of cell cycle progression. RESULTS: APE treatment caused arrest in the M phase, as indicated by the increase in p-HH3 (ser10)-positive cells. By immunocytochemistry, we found a significant increase in abnormal mitoses and multipolar mitotic spindle formation after APE treatment. Time-lapse analysis confirmed that the APE-treated GB cells were unable to correctly complete the mitosis. The modulated expression of SIRT2 and acetylated tubulin in APE-treated cells provides new insights into the mechanisms of altered mitotic progression in both GB cell lines. CONCLUSIONS: Our data show that the M2 agonist increases aberrant mitosis in GB cell lines. These results strengthen the idea of considering M2 acetylcholine receptors a novel promising therapeutic target for the glioblastoma treatment.

Sox2-dependent maintenance of mouse oligodendroglioma involves the Sox2-mediated downregulation of Cdkn2b, Ebf1, Zfp423, and Hey2.

Cancer stem cells (CSC) are essential for tumorigenesis. The transcription factor Sox2 is overexpressed in brain gliomas, and is essential to maintain CSC. In mouse high-grade glioma pHGG cells in culture, Sox2 deletion causes cell proliferation arrest and inability to reform tumors after transplantation in vivo; in Sox2-deleted cells, 134 genes are derepressed. To identify genes mediating Sox2 deletion effects, we overexpressed into pHGG cells nine among the most derepressed genes, and identified four genes, Ebf1, Hey2, Zfp423, and Cdkn2b, that strongly reduced cell proliferation in vitro and brain tumorigenesis in vivo. CRISPR/Cas9 mutagenesis of each gene, individually or in combination (Ebf1 + Cdkn2b), significantly antagonized the proliferation arrest caused by Sox2 deletion. The same genes also repressed clonogenicity in primary human glioblastoma-derived CSC-like lines. These experiments identify a network of critical tumor suppressive Sox2-targets whose inhibition by Sox2 is involved in glioma CSC maintenance, defining new potential therapeutic targets.

Cross Interaction between M2 Muscarinic Receptor and Notch1/EGFR Pathway in Human Glioblastoma Cancer Stem Cells: Effects on Cell Cycle Progression and Survival.

Glioblastomas (GBM) are the most aggressive form of primary brain tumors in humans. A key feature of malignant gliomas is their cellular heterogeneity. In particular, the presence of an undifferentiated cell population of defined Glioblastoma Stem cells (GSCs) was reported. Increased expression of anti-apoptotic and chemo-resistance genes in GCSs subpopulation favors their high resistance to a broad spectrum of drugs. Our previous studies showed the ability of M2 muscarinic receptors to negatively modulate the cell growth in GBM cell lines and in the GSCs. The aim of this study was to better characterize the inhibitory effects of M2 receptors on cell proliferation and survival in GSCs and investigate the molecular mechanisms underlying the M2-mediated cell proliferation arrest and decreased survival. Moreover, we also evaluated the ability of M2 receptors to interfere with Notch1 and EGFR pathways, whose activation promotes GSCs proliferation. Our data demonstrate that M2 receptors activation impairs cell cycle progression and survival in the primary GSC lines analyzed (GB7 and GB8). Moreover, we also demonstrated the ability of M2 receptor to inhibit Notch1 and EGFR expression, highlighting a molecular interaction between M2 receptor and the Notch-1/EGFR pathways also in GSCs.

Glioblastoma models driven by different mutations converge to the proneural subtype.

The need of reliable syngeneic animal models for gliomas has been addressed in the last decades by reproducing genetic alterations typical of human glioblastoma in the mouse. Since different alterations underlie different molecular glioblastoma subtypes it is commonly expected that tumors induced by specific alterations represent models of the corresponding subtypes. We tested this assumption by a multilevel analysis ranging from a detailed histopathological analysis to a genome-wide expression profiling by microarray and RNA-seq on gliomas induced by two distinct molecular alterations: the overstimulation of the PDGF- and the EGF- pathways. These alterations are landmarks of proneural and classical glioblastoma subtypes respectively. However, our results consistently showed a strong similarity between the two glioma models. The expression profiles of both models converged toward a signature typical of oligodendrocyte progenitor cells, regardless the wide differentiative potential of the cell of origin. A classification based on similarity with human gliomas profiles revealed that both models belong to the proneural subtype. Our results highlight that reproducing a molecular alteration specific of a glioblastoma subtype not necessarily generates a tumor model recapitulating such subtype.

Cdh4 Down-Regulation Impairs in Vivo Infiltration and Malignancy in Patients Derived Glioblastoma Cells.

The high invasive phenotype of glioblastoma is one of the main causes of therapy inefficacy and tumor relapse. Cell adhesion molecules of the cadherin family are involved in cell migration and are known as master regulators of epithelial tumor invasiveness, but their role in glioblastoma is less understood. In particular, we recently demonstrated, in the syngeneic murine model, the occurrence of a previously undescribed cadherin switch between Cdh2 and Cdh4 during gliomagenesis, which is necessary for the acquisition of the highly infiltrative and tumorigenic phenotype of these cells. In the present study, we tested the role of Cdh4 in human gliomas. Our results on patient-derived glioma cells demonstrate a positive correlation between Cdh4 expression levels and the loss of cell-cell contact inhibition of proliferation controls that allows cells to proliferate over confluence. Moreover, the silencing of Cdh4 by artificial microRNAs induced a decrease in the infiltrative ability of human glioma cells both in vitro and in vivo. More strikingly, Cdh4 silencing induced an impairment of the tumorigenic potential of these cells after orthotopic transplantation in immunodeficient mice. Overall, we conclude that in human glioblastoma, Cdh4 can also actively contribute in regulating cell invasiveness and malignancy.

Eradication of glioblastoma by immuno-virotherapy with a retargeted oncolytic HSV in a preclinical model.

Oncolytic herpes simplex viruses are proving to be effective in clinical trials against a number of cancers. Here, R-115, an oncolytic herpes simplex virus retargeted to human erbB-2, fully virulent in its target cells, and armed with murine interleukin-12 was evaluated in a murine model of glioblastoma. We show that a single R-115 injection in established tumors resulted, in about 30% of animals, in the complete eradication of the tumor, otherwise invariably lethal. The treatment also induced a significant improvement in the overall median survival time of mice and a resistance to recurrence from the same neoplasia. Such a high degree of protection was unprecedented; it was not observed before following treatments with the commonly used, mutated/attenuated oncolytic viruses. This is the first study providing the evidence of benefits offered by a fully virulent, retargeted, and armed herpes simplex virus in the treatment of glioblastoma and paves the way for clinical translation.

Progression from low- to high-grade in a glioblastoma model reveals the pivotal role of immunoediting.

The mutual reshape of tumor and immune system cells during tumor progression is a widely accepted notion in different cancers including gliomas. The importance of this phenomenon in shaping glioma progression and the mechanisms governing it, however, are not fully elucidated. Taking advantage of a well-characterized in vivo glioma model we performed an analysis of glioma cells transcriptomes at different stages of progression and unveiled the reorganization of glioma-immune system interactions. Specifically, we show that the inability of low-grade glioma cells to orthotopically graft in syngeneic immunocompetent mice, positively correlates with the abundance of infiltrating lymphocytes in donor tumors and with a highly immunostimulatory transcriptional profile. Notably, during tumor progression glioma cells downregulate these genes and the immune infiltrate shifts towards a pro-tumorigenic phenotype. Challenging low-grade gliomas by grafting into immunodeficient hosts revealed the crucial role of the adaptive immune system in constraining glioma progression. Finally, we observed that although progression still takes place in immunodeficient mice, it is slower, likely due to a milder selection thus reinforcing the view of a pivotal role for the immune system in regulating glioma progression.

Noninvasive Monitoring of Glioma Growth in the Mouse.

Malignant gliomas are the most common and deadly primary malignant brain tumors. In vivo orthotopic models could doubtless represent an appropriate tool to test novel treatment for gliomas. However, methods commonly used to monitor the growth of glioma inside the mouse brain are time consuming and invasive. We tested the reliability of a minimally invasive procedure, based on a secreted luciferase (Gaussia luciferase), to frequently monitor the changes of glioma size. Gluc activity was evaluated from blood samples collected from the tail tip of mice twice a week, allowing to make a growth curve for the tumors. We validated the correlation between Gluc activity and tumor size by analysing the tumor after brain dissection. We found that this method is reliable for monitoring human glioma transplanted in immunodeficient mice, but it has strong limitation in immunocompetent models, where an immune response against the luciferase is developed during the first weeks after transplant.

The activation of M2 muscarinic receptor inhibits cell growth and survival in human glioblastoma cancer stem cells.

The involvement of muscarinic receptors in cancer has been reported. Recently we have demonstrated that the activation of M2 muscarinic receptors, through arecaidine propargyl ester, arrests cell proliferation and induces apoptosis in primary and established glioblastoma cell lines. Considering the inability of conventional drugs to completely counteract the growth of glioblastoma cancer stem cells (GSCs), we have investigated the effect produced by arecaidine on GSC growth and survival. The expression of M2 receptors has been analyzed in GSC cell lines derived from human biopsies. Based on the M2 receptor expression levels, we have selected two gliolastoma cell lines (GB7 and GB8). In both cell lines the treatment with arecaidine decreased GCS cell growth. GB7 cells exhibited a time- and dose-dependent decrease of cell proliferation. Moreover arecaidine caused a reduced cell survival in particular in GB8 cell line. These effects appear to be mediated by M2 receptor activation as suggested by pharmacological experiments performed in the presence of M1 and M3 preferring antagonists (pirenzepine and 4-DAMP respectively) and M2/M4 antagonist methoctramine. M2 receptor silencing by siRNA has further confirmed that the inhibition of cell growth arecaidine-induced was mediated by the M2 receptor activation. These results suggest that the M2 receptors may represent a new interesting therapeutic tool to counteract glioblastoma cancer stem cell growth and survival.

T-wave axis deviation, metabolic syndrome and estimated cardiovascular risk--in men and women of the MOLI-SANI study.

AIM: We aimed at investigating the association between T-wave axis deviation, metabolic syndrome (MetS), its components and estimated risk of cardiovascular disease (CVD) at 10 years in an adult Italian population. METHODS: 11,143 women (54 ± 11 years) and 9742 men (55 ± 11 years) were analyzed from the Moli-sani cohort, randomly recruited from the general population. MetS was defined using the ATPIII criteria. T-wave axis deviation was measured from the standard 12-lead resting electrocardiogram. CVD risk in ten years was estimated by the CUORE score. RESULTS: 29% of men and 27% of women with MetS showed borderline or abnormal T-wave as compared to 24% and 17% without MetS (p < 0.0001 for both genders). Among components of MetS, elevated waist and blood pressure were strongly associated with T-wave axis deviation, whereas glucose, HDL and triglycerides were only marginally. The odds of having borderline or abnormal T-wave axis deviation in multivariable regression analysis, was 1.38 (95% CI:1.25-1.53) in MetS men and 1.68 (95% CI:1.51-1.87) in MetS women compared to those without. Further adjustment for MetS components completely abolished the associations. Abnormal T-wave axis deviation was associated with an increased risk of CVD in 10 years in men (OR = 4.4; 95% CI:1.10-17.9). CONCLUSION: T-wave axis deviation is strongly associated with components of the MetS, in particular high waist circumference and blood pressure and with an increased CVD risk, particularly in men. ECG monitoring to identify T-wave axis deviation in obese, hypertensive or MetS subjects can be an early indicator of vascular disease and help in reducing cardiac events.

Implantation of a new mitral ring, adjustable during follow-up: a simplified technique.

Mitral valve repair for ischaemic mitral incompetence has a 10% rate of failure at ten year follow-up. Progressive annular dilation could play an important role. We have implanted the enCor(SQ)(TM) mitral valve repair system. This system can be downsized during follow-up with the appropriate activation via the lead passed through the left atrium suture line, in order to restore mitral leaflet coaptation.

Prospective, randomized clinical trial of the FloSeal matrix sealant in cardiac surgery.

BACKGROUND: Topical hemostatic agents composed of a gelatin-based matrix and thrombin have been reported to be effective, in addition to traditional means, in terminating bleeding during cardiac operations. We compared a hemostatic matrix sealant agent (FloSeal; Baxter Inc, Deerfield, IL) with alternative topical hemostatic agents in a mixed cohort of elective cardiac and thoracic aortic operations. METHODS: Following sample size calculation, in a prospective randomized study design, 209 patients were treated with FloSeal matrix sealant (FloSeal group) and 206 patients received alternative agents as topical hemostatic materials (comparison group). FloSeal is composed of a self-expandable gelatin matrix component and purified bovine thrombin. Comparisons included hemostatic patches or sponges composed of either oxidized regenerated cellulose or purified porcine skin gelatin. Study endpoints were the following: rate of successful intraoperative hemostasis (identified by cessation of bleeding) and time required for hemostasis; overall postoperative bleeding; rate of transfusion of blood products; rate of surgical revision for bleeding; postoperative morbidity; and intensive care unit stay. RESULTS: Statistically higher rates of successful hemostasis and shorter time-to-hemostasis were observed in the FloSeal group (p < 0.001 both). Time-to-event analysis confirmed this finding (p = 0.0025). Postoperative bleeding and rate of transfusion of blood products were statistically decreased in the FloSeal group (p < 0.001 both). Rates of revision for bleeding and of minor complications were not statistically different among groups in the overall cohort, but were significantly lesser in the FloSeal group if only patients with overt intraoperative bleeding are considered (p = 0.04 both). The advantages observed in the FloSeal group were not offset in patients undergoing systemic hypothermia. CONCLUSIONS: The topical hemostatic agent used in the FloSeal group is effective in terminating intraoperative bleeding as an adjunct to traditional surgical methods for stopping bleeding. Its judicious use is associated with lesser need for transfusion of blood products and rate of revision for bleeding. Its cost-utility profile should be addressed in dedicated trials.

Incidence, determinants, and prognostic value of reverse left ventricular remodelling after primary percutaneous coronary intervention: results of the Acute Myocardial Infarction Contrast Imaging (AMICI) multicenter study.

AIMS: Few data are available on the extent and prognostic value of reverse left ventricular remodelling (r-LVR) after ST-elevation acute myocardial infarction (STEMI). We sought to evaluate incidence, major determinants, and long-term clinical significance of r-LVR in a group of STEMI patients treated with primary percutaneous coronary intervention (PPCI). In particular, the role of preserved microvascular flow within the infarct zone in inducing r-LVR has been investigated. METHODS AND RESULTS: Serial echocardiograms (2DE) and myocardial contrast study were obtained within 24 h of coronary recanalization (T1) and at pre-discharge (T2) in 110 reperfused STEMI patients. Follow-up 2DE was scheduled after 6 months (T3). Two-year clinical follow-up was obtained. Reverse remodelling was defined as a reduction >10% in LV end-systolic volume (LVESV) at 6 months follow-up. r-LVR occurred in 39% of study population. At multivariable analysis, independent predictors of r-LVR were an effective microvascular reflow within the infarct zone, the in-hospital improvement of myocardial perfusion, an initial large LVESV, and a short time to reperfusion. Cox analysis identified r-LVR as the only independent predictor of 2-year event-free survival. Combined events rate was significantly higher among patients without compared to those with r-LVR (log-rank test P < 0.05). CONCLUSION: r-LVR frequently occurs in STEMI patients treated with PPCI and it is an important predictor of favourable long-term outcome. A preserved microvascular perfusion within the infarct zone is the major determinant of r-LVR.

Pseudoaneurysm after patch-free carotid bifurcation endarterectomy: a case report.

The purpose of this report is to present the case of a 59-year-old man affected by multivessel coronary artery disease and sterile pseudoaneurysm of the right carotid bifurcation presenting as a pulsating neck mass 1 month after patch-free carotid endarterectomy. The surgical approach included median sternotomy and incision parallel to the anterior margin of the right sternocleidomastoid muscle. The pseudoaneurysm was excised after control of the brachiocefalic trunk and insertion of a Pruit-Inahara shunt, and the carotid vessels were directly sutured without using any prosthetic or autologous material. Concomitant coronary bypass grafting was performed. The postoperative course was uneventful and there was no evidence of recurrence of pseudoaneurysm at 6-month follow-up. On the basis of this experience and of pertinent literature, the options for the management of such rare entities are discussed.

Evaluation of less invasive method for saphenous vein harvest in patients with type II diabetes.

BACKGROUND: Diabetes mellitus is an established risk factor for leg wound healing complications after great saphenous vein harvest. Leg healing complications occur in 1-25% of coronary artery bypass graft patients, and are often underestimated. PATIENTS AND METHODS: The records of 230 patients enrolled in a prospective trial to evaluate a minimally invasive approach compared with conventional longitudinal harvest were reviewed. Of 100 patients with diabetes, 49 had undergone minimally invasive harvest (group A). Forty-nine patients from the pool without diabetes who underwent minimally invasive harvest were selected using propensity scoring analysis (group B), and 46 diabetic patients operated using a conventional technique (group C) were matched to group A patients. Ninety-five patients with and 49 without diabetes finally entered the study. The quality of leg wound healing was quantified by ASEPSIS score by two independent surgeons in a blinded manner. The occurrence of complications was compared between groups. RESULTS: Fewer leg wound healing complications occurred in diabetic patients in the minimally invasive compared with the conventional group (P < 0.0001). Rates of complications were comparable among diabetic and non-diabetic patients operated using the minimally invasive technique. The intraoperative flow of vein grafts obtained by the minimally invasive technique was comparable to that of veins harvested using the conventional technique. CONCLUSION: These data suggest that postoperative leg wound morbidity can be significantly attenuated by the adoption of a less invasive approach, even in high-risk patients with diabetes. The advantages are not offset by the co-existence of other cardiovascular risk factors or by a long history of diabetes.

Open clip-free radial artery harvesting with the harmonic shears.

BACKGROUND: The Ultracision Harmonic Scalpel is associated with several advantages in radial artery (RA) harvesting. It allows fewer hemostatic clips to close the collateral branches, less thermal injury of the conduit, and reduced time of harvesting in comparison with the conventional RA harvesting technique with electrocautery and hemostatic clips. We recently started open RA harvesting with the harmonic shears (HSH). In this study, we aimed at evaluating the feasibility of this simplified ultrasonically activated harvesting technique, and report the results of RA harvesting with HSH. METHODS: The RA harvesting with HSH was performed in 20 patients operated on for myocardial revascularization from July 2004 to December 2005. RESULTS: The harvest of the RA was completed in little time, without any complication. Neither bleeding from the collateral branches nor spasm alongside the entire length of the RA was observed. No bleeding occurred from the muscles of the forearm. CONCLUSION: This technique of RA harvesting with HSH is impressive in terms of short time of harvest, complete absence of clips for the collateral branches, and no thermal injury of the conduit.