Up-regulation of HIF-1α is associated with neuroprotective effects of agmatine against rotenone-induced toxicity in differentiated SH-SY5Y cells.

Agmatine, a metabolite generated by arginine decarboxylation, has been reported as neuromodulator and neuroactive substance. Several findings suggest that agmatine displays neuroprotective effects in several models of neurodegenerative disorders, such as Parkinson's disease (PD). It has been hypothesized that biogenic amines may be involved in neuroprotection by scavenging oxygen radicals, thus preventing the generation of oxidative stress. Mitochondrial dysfunction, that leads to a reduction of oxygen consumption, followed by activation of prolyl hydroxylase and decrease of hypoxia-inducible factor 1 alpha (HIF-1α) levels, has been demonstrated to play a role in PD pathogenesis. Using rotenone-treated differentiated SH-SY5Y cells as the in vitro PD model, we here investigated the molecular mechanisms underlying agmatine neuroprotective effects. Our results showed that the preliminary addition of agmatine induces HIF-1α activation, and prevents the rotenone-induced production of free radical species, and the activation of apoptotic pathways by inhibiting mitochondrial membrane potential decrease and caspase 3 as well as cytochrome c increase. Notably, these effects are mediated by HIF-1α, as indicated by experiments using a HIF-1α inhibitor. The present findings suggest that the treatment with agmatine is able to counteract the neuronal cell injury evoked by mitochondrial toxins.

Hypoxia-Dependent Expression of TG2 Isoforms in Neuroblastoma Cells as Consequence of Different MYCN Amplification Status.

Transglutaminase 2 (TG2) is a multifunctional enzyme and two isoforms, TG2-L and TG2-S, exerting opposite effects in the regulation of cell death and survival, have been revealed in cancer tissues. Notably, in cancer cells a hypoxic environment may stimulate tumor growth, invasion and metastasis. Here we aimed to characterize the role of TG2 isoforms in neuroblastoma cell fate under hypoxic conditions. The mRNA levels of TG2 isoforms, hypoxia-inducible factor (HIF)-1α, p16, cyclin D1 and B1, as well as markers of cell proliferation/death, DNA damage, and cell cycle were examined in SH-SY5Y (non-MYCN-amplified) and IMR-32 (MYCN-amplified) neuroblastoma cells in hypoxia/reoxygenation conditions. The exposure to hypoxia induced the up-regulation of HIF-1α in both cell lines. Hypoxic conditions caused the up-regulation of TG2-S and the reduction of cell viability/proliferation associated with DNA damage in SH-SY5Y cells, while in IMR-32 did not produce DNA damage, and increased the levels of both TG2 isoforms and proliferation markers. Different cell response to hypoxia can be mediated by TG2 isoforms in function of MYCN amplification status. A better understanding of the role of TG2 isoforms in neuroblastoma may open new venues in a diagnostic and therapeutic perspective.

Neuromuscular Plasticity in a Mouse Neurotoxic Model of Spinal Motoneuronal Loss.

Despite the relevant research efforts, the causes of amyotrophic lateral sclerosis (ALS) are still unknown and no effective cure is available. Many authors suggest that ALS is a multi-system disease caused by a network failure instead of a cell-autonomous pathology restricted to motoneurons. Although motoneuronal loss is the critical hallmark of ALS given their specific vulnerability, other cell populations, including muscle and glial cells, are involved in disease onset and progression, but unraveling their specific role and crosstalk requires further investigation. In particular, little is known about the plastic changes of the degenerating motor system. These spontaneous compensatory processes are unable to halt the disease progression, but their elucidation and possible use as a therapeutic target represents an important aim of ALS research. Genetic animal models of disease represent useful tools to validate proven hypotheses or to test potential therapies, and the conception of novel hypotheses about ALS causes or the study of pathogenic mechanisms may be advantaged by the use of relatively simple in vivo models recapitulating specific aspects of the disease, thus avoiding the inclusion of too many confounding factors in an experimental setting. Here, we used a neurotoxic model of spinal motoneuron depletion induced by injection of cholera toxin-B saporin in the gastrocnemius muscle to investigate the possible occurrence of compensatory changes in both the muscle and spinal cord. The results showed that, following the lesion, the skeletal muscle became atrophic and displayed electromyographic activity similar to that observed in ALS patients. Moreover, the changes in muscle fiber morphology were different from that observed in ALS models, thus suggesting that some muscular effects of disease may be primary effects instead of being simply caused by denervation. Notably, we found plastic changes in the surviving motoneurons that can produce a functional restoration probably similar to the compensatory changes occurring in disease. These changes could be at least partially driven by glutamatergic signaling, and astrocytes contacting the surviving motoneurons may support this process.

Clobetasol Modulates Adult Neural Stem Cell Growth via Canonical Hedgehog Pathway Activation.

Sonic hedgehog (Shh) signaling is a key pathway within the central nervous system (CNS), during both development and adulthood, and its activation via the 7-transmembrane protein Smoothened (Smo) may promote neuroprotection and restoration during neurodegenerative disorders. Shh signaling may also be activated by selected glucocorticoids such as clobetasol, fluocinonide and fluticasone, which therefore act as Smo agonists and hold potential utility for regenerative medicine. However, despite its potential role in neurodegenerative diseases, the impact of Smo-modulation induced by these glucocorticoids on adult neural stem cells (NSCs) and the underlying signaling mechanisms are not yet fully elucidated. The aim of the present study was to evaluate the effects of Smo agonists (i.e., purmorphamine) and antagonists (i.e., cyclopamine) as well as of glucocorticoids (i.e., clobetasol, fluocinonide and fluticasone) on NSCs in terms of proliferation and clonal expansion. Purmorphamine treatment significantly increased NSC proliferation and clonal expansion via GLI-Kruppel family member 1 (Gli1) nuclear translocation and such effects were prevented by cyclopamine co-treatment. Clobetasol treatment exhibited an equivalent pharmacological effect. Moreover, cellular thermal shift assay suggested that clobetasol induces the canonical Smo-dependent activation of Shh signaling, as confirmed by Gli1 nuclear translocation and also by cyclopamine co-treatment, which abolished these effects. Finally, fluocinonide and fluticasone as well as control glucocorticoids (i.e., prednisone, corticosterone and dexamethasone) showed no significant effects on NSCs proliferation and clonal expansion. In conclusion, our data suggest that Shh may represent a druggable target system to drive neuroprotection and promote restorative therapies.

Transglutaminase 2 is involved in amyloid-beta1-42-induced pro-inflammatory activation via AP1/JNK signalling pathways in THP-1 monocytes.

Deposition of amyloid-beta (Aß) peptides has been shown to induce the release of inflammatory factors by activated microglia and brain infiltrating monocytes/macrophages. Interestingly, the enzyme transglutaminase 2 (TG2) has been shown to play a key role in neuroinflammation and regulation of transcription factors involved in immunomodulation. In this study, we aimed to better elucidate the mechanisms underlying TG2 involvement in the pro-inflammatory signaling pathway activated by fibrillar Aß1-42 in THP-1 monocytes. Cell exposure for 24 h to 500 nM Aß1-42, induced the up-regulation of CD14, CD16, and TG2, suggesting THP-1 cell functional activation. Aß1-42 also increased the production of reactive oxygen species, that was reduced by the pre-incubation with genistein (25 µg/ml), a soy isoflavone with antioxidant properties. Moreover, IL-1ß and IL-6 mRNA transcript and protein levels were eightfold increased in Aß1-42-treated THP-1 monocytes. Interestingly, these effects were significantly reduced by R283 (~45%), a specific inhibitor of TG activity, and genistein (~40%). Aß1-42 induced the activation of p54/p46 JNK, as well as ERK 1/2 at a lower extent. The inactivation of ERK1/2 signalling pathway, but not JNK, by either genistein or U0126, a MEK1/2 inhibitor, was not able to blunt Aß1-42-induced TG2 up-regulation, that, instead, was significantly reduced by R283. Aß1-42 also induced AP-1 activation that was not significantly affected by genistein or U0126, while was strongly reduced by R283. Our preliminary findings first suggest that TG2 up-regulation is involved in the pro-inflammatory activation of THP-1 monocytes induced by Aß1-42 via AP1/JNK signalling pathways.

Transglutaminase 2 up-regulation is associated with RANKL/OPG pathway in cultured HPDL cells and THP-1-differentiated macrophages.

Recent evidence emphasized that transglutaminase 2 (TG2), a protein cross-linking enzyme, may play a role in the early phase of inflammation. High levels of TG2 have been associated with the constitutive activation of nuclear factor-kappa B (NF-κB) that is considered the main regulator of inflammation. In this context, the receptor activator of NF-kappa B ligand (RANKL) and receptor activator of NF-κB have extensive functions in the regulation of cytokine secretion associated with different pathological conditions. The human periodontal ligament (HPDL) cells, which express and secrete osteoprotegerin (OPG) and RANKL, represent an useful "ex vivo" model for monitoring cell response in inflammatory microenvironments, such as periodontitis-dependent tissue response. Thus, we evaluated TG2 expression and alterations in RANKL/OPG ratio occurring in cultured HPDL cells. The HPDL cells were obtained from patients with chronic periodontitis (CP) and healthy subjects. We observed the up-regulation of some inflammatory markers, such as IL-6, TNF-α, and HMGB-1, and at the same time an increase in TG2 mRNA levels in HPDL cells from CP patients compared with healthy subjects. We found a positive correlation between RANKL/OPG ratio and TG2 mRNA levels in HPDL cells from CP patients. In the parallel experiments, we demonstrated that TG2 inhibition reduced RANKL expression in both HPDL cells from CP patients and monocytes differentiated to macrophages by tetradecanoyl phorbol acetate treatment. Given the RANKL key role in NF-κB pathway and the observed up-regulation of pro-inflammatory cytokines, our data suggest that TG2 may be involved in molecular mechanisms of inflammatory response occurring in periodontal disease.

Neuroprotective effects of phloretin and its glycosylated derivative on rotenone-induced toxicity in human SH-SY5Y neuronal-like cells.

Phloretin and phlorizin are the two strong natural antioxidants whose biological and pharmacological applications are rapidly growing in different human pathological conditions. The neuroprotective activity of the two flavonoids has been analyzed on cell culture of neuroblastoma cells. The neuroprotective activity of the two flavonoids has been analyzed on cell culture of neuroblastoma cells and evaluated by testing cell vitality, mitochondrial transmembrane potential and ROS production, antioxidant enzymes detection, activation of caspase 3, DNA damage, protein carbonylation, lipid peroxidation, and superoxide anion scavenging activity. Incubation of cells with rotenone caused cell death and significant increase in intracellular reactive oxygen species, activation of caspase 3, and variation in mitochondrial transmembrane potential. Although, rotenone exposure caused a significant increase of antioxidant enzymes, high levels of lipid peroxidation were also observed. Phloretin or phlorizin, at micromolar concentration, reduced rotenone-induced cell death by scavenging ability against superoxide anion radical, one of the main effectors of rotenone toxicity at level of mitochondrial respiratory chain complex I. Under our experimental conditions, a reduction of the intracellular ROS levels with consequent normalization of the aforementioned antioxidant enzymes occurred. Concomitantly, we observed the inhibition of caspase 3 activity and DNA damage. This study shows the promising neuroprotective ability of the two dihydrochalcones able to protect human differentiated neuroblastoma cells (commonly used as model of Parkinson's disease) from injury induced by rotenone, actively scavenging ROS, normalizing mitochondrial transmembrane potential and consequently avoiding energy depletion. © 2017 BioFactors, 43(4):549-557, 2017.

Non-Seminomatous Germ Cell Tumor Metastasis to the Jaw: An Imaging Case Report.

Testicular cancer typically spreads to the lymph nodes, but hematogenous dissemination to distant organs can also occur. Bone metastasis is uncommon and is thought to be a poor prognostic indicator. Jaw metastasis is exceedingly rare but is of great clinical significance, since it may be the first sign of an occult testicular cancer or the first evidence of relapse of a known tumor. Herein, we report the first case describing the imaging and clinical findings of a non-seminomatous germ cell tumor with jaw metastasis at diagnosis.

Giant leiomyoma of the retzius space: a case report.

Extrauterine leiomyoma is a very rare clinical condition; we report a case of leiomyoma of the Retzius space in a 49-year-old women who suffered for two years from bladder voiding symptoms characterized by dysuria, feeling of incomplete emptying, and pelvic pain. Clinical evaluation and abdominal and transvaginal ultrasound suggested the presence of a voluminous (about 10 cm in diameter) fibromyoma of the anterior uterus surface. The urodynamic evaluation demonstrated the presence of bladder outlet obstruction (voiding pressure greater than 20 cm H2O and maximum flow rate less than 12 mL/s) with a postvoiding urine residual equal to 80 mL; moreover, the presence of cystocele and urethral stricture was ruled out performing clinical evaluation, cystography, and cystourethroscopy. The patient underwent laparotomy to remove the uterine fibromyoma. Intraoperatively, a voluminous soft mass arising from the Retzius space was found; it was firmly adhered to the uterus with obliteration of vesicouterine pouch owing to severe adhesion to the anterior surface of uterus. The tumour was isolated, enucleated from the prevesical space, and removed; moreover, the patient became asymptomatic after surgery. In conclusion, leiomyoma of the Retzius space is a very rare benign tumour that should be considered in the presence of severe bladder voiding symptoms.

Reflexiones sobre la caries de la infancia temprana severa / Reflections on early childhood caries

Introducción: el incremento de las visitas de infantes en nuestras respectivas consultas (menores de tres años) con caries de la infancia temprana severa (CIT-S) en estos últimos años es el origen del presente artíuculo de análisis. Objetivo: describir las características de la (CIT-S) plantear hipótesis sobre su presentación clínica; así como agrupar y resumir los factores de riesgo de este particular grupo etéreo. Conclusiones: mientras mejor comprendamos todos los factores de riesgo que participan en esta enfermedad tan multifactorial y prevalente en la infancia, mejores pautas podremos ofrecer a los padres de nuestros pacientes, basadas en el riesgo individual del infante

Introduction: increased visits in our private practice of infants (under 3 years) with severe early childhood caries (CIT-S) in the recent years is the source of this analysis article. Aim: describe the characteristics of the early childhood caries, hypothesize about its clinical presentation; as well as group and summarize the risk factors of this particular age group. Conclusions: the better we understand all the risk factors involved in this disease as multifactorial and prevalent in childhood, best guidelines we offer parents of our patients, based on the individual risk of the infant.