CELL DAMAGE INDUCED BY LYSOSOMAL IMPAIRMENT: STUDY OF THE ROLE OF PLASMA MEMBRANE SPHINGOLIPIDS

Lysosomes are the principal site of the catabolism of sphingolipids, a class of bioactive lipids mainly associated with the external leaflet of cell plasma membrane. Several lines of evidence support a direct correlation between modifications in sphingolipid pattern and the activation of specific signaling pathways, including apoptosis and autophagy. Loss-of-function mutations in genes coding for lysosomal enzymes involved in sphingolipid catabolism result in severe clinical manifestations called sphingolipidoses. These pathologies belong to the group of Lysosomal Storage Diseases and are characterized by the accumulation of undegraded materials leading to lysosomal impairment and consequent cell damage. Until now, the molecular mechanisms by which the perturbation of lysosomal homeostasis affects cell functionality and viability are unknown. To investigate this issue, I used an artificial in vitro model of lysosomal impairment obtained by loading human fibroblasts with 88 mM sucrose for 14 days. In these experimental conditions, the absence of invertase induces sucrose accumulation into lysosomes. I found that sucrose loaded fibroblasts are characterized by a growth slowdown and by the activation of both apoptosis and autophagy. By RNA-sequencing, approximately a thousand of genes were found to be dysregulated after sucrose loading. In particular, 56 cell cycle-related genes are downregulated, whereas 37 lysosomal-related genes are upregulated. Using biochemical approaches, I found that sucrose loading activates lysosomal biogenesis although sucrose storage inhibits lysosomal functionality. In particular, in sucrose loaded cells lipid catabolism is blocked and complex lipids, such as phospholipids, cholesterol, glycosphingolipids, and gangliosides are accumulated. Moreover, I found that sucrose loading induces the nuclear translocation of the Transcription Factor EB (TFEB), a master-gene regulator of lysosomal function, which in turn promotes the increased fusion between lysosomes and the plasma membrane. This last event leads to higher levels of sphingolipid hydrolases at the cell surface resulting in the alteration of the plasma membrane sphingolipid composition and the consequent ectopic production of pro-apoptotic and pro-autophagic ceramide. Interestingly, in sucrose loaded fibroblasts the blocking of glycosphingolipid hydrolysis at the plasma membrane results in a reduction of autophagy and apoptosis. Similar results were also obtained in response to sphingomyelin accumulation in Niemann-Pick Type A disease (NPA). NPA is a sphingolipidosis caused by acid sphingomyelinase deficiency which leads to sphingomyelin storage. Interestingly, using NPA-derived human fibroblasts loaded with 50 \ub5M exogenous sphingomyelin for 30 days, I found that the lysosomal impairment caused by sphingomyelin accumulation activates the same molecular pathways described in healthy fibroblasts subjected to sucrose loading. A pathogenic role of TFEB has also been suggested by biochemical analysis on brains from Acid Sphingomyelinase Knockout (ASMKO) mice. In fact, ASMKO mouse brains are characterized by TFEB nuclear translocation, increased lysosomal biogenesis, increased glycohydrolytic activities and onset of apoptosis and autophagy. Collectively, these data suggest the existence of a cross-talk among lysosomes and the cell plasma membrane. In this context, the lysosomal impairment caused by the accumulation of uncatabolized substrates leads to an altered composition of plasma membrane sphingolipids resulting in the ectopic production of ceramide which in turn is responsible for the onset of cell damage

Genetic basis for variation in plasma IL-18 levels in persons with chronic hepatitis C virus and human immunodeficiency virus-1 infections

Inflammasomes are multi-protein complexes integrating pathogen-triggered signaling leading to the generation of pro-inflammatory cytokines including interleukin-18 (IL-18). Hepatitis C virus (HCV) and human immunodeficiency virus (HIV) infections are associated with elevated IL-18, suggesting inflammasome activation. However, there is marked person-to-person variation in the inflammasome response to HCV and HIV. We hypothesized that host genetics may explain this variation. To test this, we analyzed the associations of plasma IL-18 levels and polymorphisms in 10 genes in the inflammasome cascade. About 1538 participants with active HIV and/or HCV infection in three ancestry groups are included. Samples were genotyped using the Illumina Omni 1-quad and Omni 2.5 arrays. Linear regression analyses were performed to test the association of variants with log IL-18 including HCV and HIV infection status, and HIV RNA in each ancestry group and then meta-analyzed. Eleven highly correlated single-nucleotide polymorphisms (r²=0.98–1) in the IL-18-BCO2 region were significantly associated with log IL-18; each T allele of rs80011693 confers a decrease of 0.06 log pg ml⁻¹ of IL-18 after adjusting for covariates (rs80011693; rs111311302 β=−0.06, P-value=2.7 × 10⁻⁴). In conclusion, genetic variation in IL-18 is associated with IL-18 production in response to HIV and HCV infection, and may explain variability in the inflammatory outcomes of chronic viral infections

Mitochondrial Dysregulation and Impaired Autophagy in iPSC-Derived Dopaminergic Neurons of Multiple System Atrophy

Multiple system atrophy (MSA) is a progressive neurodegenerative disease that affects several areas of the CNS, whose pathogenesis is still widely unclear and for which an effective treatment is lacking. We have generated induced pluripotent stem cell-derived dopaminergic neurons from four MSA patients and four healthy controls and from two monozygotic twins discordant for the disease. In this model, we have demonstrated an aberrant autophagic flow and a mitochondrial dysregulation involving respiratory chain activity, mitochondrial content, and CoQ10 biosynthesis. These defective mechanisms may contribute to the onset of the disease, representing potential therapeutic targets. Monzio Compagnoni et al. present an iPSC-based neuronal in vitro model of multiple system atrophy. Patients' dopaminergic neurons display a dysregulation of mitochondrial functioning and autophagy, suggesting new hints for the comprehension of the pathogenesis of the disease

A comparison of two implants with conical vs internal hex connections: 1-year post-loading results from a multicentre, randomised controlled trial

Purpose: To compare the clinical and radiological outcomes of identical implants with conical or internal hex connections. Methods: A total of 90 patients with partial edentulism requiring one implant-supported prosthesis were randomly allocated in two equal groups (n = 45) to receive either implants with a conical connection or implants of the same type, but with an internal hex connection at three centres. Patients were followed for 1 year after loading. Outcome measures were implant failures, any complication and marginal bone level changes. Results: One patient (2.2%) belonging to the internal hex group dropped out. One implant (2.2%) failed in the conical group. There were no statistically significant differences in implant failures between the two groups (2.2% vs. 0%, difference 2.2; 95% CI: -1.3; 5.7; P = 0.315). Two complications occurred in the conical group and two in the internal hex group (P = 1.000, difference 0.00, 95% CI: -3.1; 3.1). The 12-month peri-implant bone resorption was similar in both groups: 0.56 ± 0.53 mm (95% CI 0.03; 1.09) in the conical group and 0.60 ± 0.62 mm (95% CI 0.02; 1.22) in the internal hex group (difference = 0.04 ± 0.55, 95% CI: -0.51; 0.59, P = 0.745). Conclusions: Within the limitation of this study, preliminary short-term data (1 year post-loading) did not show any statistical differences between the two internal connection types, therefore clinicians could choose whichever connection they prefer

Sphingosine-1-P and its plasma membrane receptors in human glioma cells

Glioblastoma multiforme (GBMs), the most frequent and deadly brain tumors in humans, are characterized by extended invasiveness and cell growth. Different sphingolipid metabolites, such as sphingosine-1-phosphate (S1P), have emerged as active mediators in the complex network of signaling pathways involved in the control of physiological and pathological cell behavior (1). Increasing evidence supports that S1P is implicated in sustaining cell invasiveness. On the other side, various growth factor receptors, such as EGFR, are frequently mutated and/or overexpressed in GBMs (2). Very importantly, S1P is able to regulate EGFR expression in lung adenocarcinoma and rat vascular smooth muscle cells suggesting the existence of a cross-talk between the S1P axis and growth factor signaling pathways induced by EGF in tumors. We investigated the crosstalk between S1P and the EGF/EGFR pathways, focusing on its role in glioma invasiveness. We used U87MG human GBMs cell line overexpressing EGF receptor (EGFR+). EGFR+ cells are characterized by increased levels of extracellular S1P and an higher expression of the active SK-1 form (phosphorylated SK-1). These cells showed increased ability to invade into Matrigel. The high chemioinvasion ability as well as spheroids sprouting were significantly inhibited in EGFR+ cells treated with SK inhibitors, or with S-FTY720-vinylphosphonate, the antagonist of S1P receptors. Moreover, we found that S1P added to the cell medium maintained the ability to drastically increase invasion in EGFR+ cells treated with SK inhibitors. At variance, S1P did not induced invasion over the basal values when glioma cells were treated with FTY720. Altogether our data strongly suggest that increased S1P secretion and signalling associated with EGFR overexpression/iperactivation play an important role in EGFR+ glioblastoma invasiveness by enhancing the invasion potential of GBM. References 1. Giussani et al. (2014) Int. J. Mol. Sci., 15, 4356 2. Zahonero et al (2014) Cell Mol Life Sci. 71, 346

Crosstalk between S1P and EGFR signalling pathways in human glioma cell invasiveness

Glioblastoma multiforme (GBMs), the most frequent and deadly brain tumor in humans, are characterized by extended invasiveness and cell growth. Further elucidation of signaling pathways responsible for the malignant phenotype and resistance to therapy will therefore represent a significant advance in GBM treatment and prognosis. Different sphingolipid metabolites, such as ceramide (Cer), and sphingosine-1-phosphate (S1P), have emerged as active mediators in the complex network of signaling pathways involved in the control of physiological and pathological cell behaviour. Increasing evidence supports that S1P is implicated in sustaining cell survival and invasiveness. On the other side, various growth factor receptors are frequently mutated and/or overexpressed in GBMs. Very importantly, S1P is able to regulate EGFR expression in lung adenocarcinoma and rat vascular smooth muscle cells. These results suggest the existence of a cross-talk between the S1P axis and growth factor signaling pathways induced by EGF in tumors. We investigated the crosstalk between S1P and the EGF/EGFR pathways, focusing on its role in glioma tumor invasiveness. We used U87MG human GBMs cell line overexpressing EGF receptor (EGFR+) or empty vector (EGFR-). EGFR+ cells are characterized by increased levels of extracellular S1P, a slightly higher sphingosine kinase-1 (SK-1) activity, and an higher expression of the active SK-1 form (phosphorylated SK-1). These cells showed increased ability to invade into Matrigel in comparison with EGFR- cells. The high chemioinvasion ability as well as spheroids sprouting were significantly inhibited in EGFR+ cells treated with SK inhibitors (SKI and PF543), or with S-FTY720-vinylphosphonate, the antagonist of S1P1\u20133-5 receptors. Moreover, we found that S1P added to the cell medium maintained the ability to drastically increase invasion in EGFR+ cells treated with SKI or PF543. At variance, S1P did not induced invasion over the basal values when glioma cells were treated with FTY720. Altogether our data strongly suggest that increased S1P secretion and signalling associated with EGFR overexpression/iperactivation play an important role in EGFR+ glioblastoma invasiveness by enhancing the invasion potential of GBM. This study was supported by Piano di sostegno alla ricerca BIOMETRA \u2013 Linea B (grant 15-6-3003005-31) to PG

Exploring the link between ceramide and ionizing radiation

The aim of radiotherapy is to eradicate cancer cells with ionizing radiation; tumor cell death following irradiation can be induced by several signaling pathways, most of which are triggered as a consequence of DNA damage, the primary and major relevant cell response to radiation. Several lines of evidence demonstrated that ceramide, a crucial sensor and/or effector of different signalling pathways promoting cell cycle arrest, death and differentiation, is directly involved in the molecular mechanisms underlying cellular response to irradiation. Most of the studies strongly support a direct relationship between ceramide accumulation and radiation-induced cell death, mainly apoptosis; for this reason, defining the contribution of the multiple metabolic pathways leading to ceramide formation and the causes of its dysregulated metabolism represent the main goal in order to elucidate the ceramide-mediated signaling in radiotherapy. In this review, we summarize the current knowledge concerning the different routes leading to ceramide accumulation in radiation-induced cell response with particular regard to the role of the enzymes involved in both ceramide neogenesis and catabolism. Emphasis is placed on sphingolipid breakdown as mechanism of ceramide generation activated following cell irradiation; the functional relevance of this pathway, and the role of glycosphingolipid glycohydrolases as direct targets of ionizing radiation are also discussed. These new findings add a further attractive point of investigation to better define the complex interplay between sphingolipid metabolism and radiation therapy