Extracellular sphingosine-1-phosphate : a novel actor in human glioblastoma stem cell survival

Glioblastomas are the most frequent and aggressive intracranial neoplasms in humans, and despite advances and the introduction of the alkylating agent temozolomide in therapy have improved patient survival, resistance mechanisms limit benefits. Recent studies support that glioblastoma stem-like cells (GSCs), a cell subpopulation within the tumour, are involved in the aberrant expansion and therapy resistance properties of glioblastomas, through still unclear mechanisms. Emerging evidence suggests that sphingosine-1-phosphate (S1P) a potent onco-promoter able to act as extracellular signal, favours malignant and chemoresistance properties in GSCs. Notwithstanding, the origin of S1P in the GSC environment remains unknown. We investigated S1P metabolism, release, and role in cell survival properties of GSCs isolated from either U87-MG cell line or a primary culture of human glioblastoma. We show that both GSC models, grown as neurospheres and expressing GSC markers, are resistant to temozolomide, despite not expressing the DNA repair protein MGMT, a major contributor to temozolomide-resistance. Pulse experiments with labelled sphingosine revealed that both GSC types are able to rapidly phosphorylate the long-chain base, and that the newly produced S1P is efficiently degraded. Of relevance, we found that S1P was present in GSC extracellular medium, its level being significantly higher than in U87-MG cells, and that the extracellular/intracellular ratio of S1P was about ten-fold higher in GSCs. The activity of sphingosine kinases was undetectable in GSC media, suggesting that mechanisms of S1P transport to the extracellular environment are constitutive in GSCs. In addition we found that an inhibitor of S1P biosynthesis made GSCs sensitive to temozolomide (TMZ), and that exogenous S1P reverted this effect, thus involving extracellular S1P as a GSC survival signal in TMZ resistance. Altogether our data implicate for the first time GSCs as a pivotal source of extracellular S1P, which might act as an autocrine/paracrine signal contributing to their malignant properties

The yeast P5 type ATPase, Spf1, regulates manganese transport into the endoplasmic reticulum

The endoplasmic reticulum (ER) is a large, multifunctional and essential organelle. Despite intense research, the function of more than a third of ER proteins remains unknown even in the well-studied model organism Saccharomyces cerevisiae. One such protein is Spf1, which is a highly conserved, ER localized, putative P-type ATPase. Deletion of SPF1 causes a wide variety of phenotypes including severe ER stress suggesting that this protein is essential for the normal function of the ER. The closest homologue of Spf1 is the vacuolar P-type ATPase Ypk9 that influences Mn2+ homeostasis. However in vitro reconstitution assays with Spf1 have not yielded insight into its transport specificity. Here we took an in vivo approach to detect the direct and indirect effects of deleting SPF1. We found a specific reduction in the luminal concentration of Mn2+ in ∆spf1 cells and an increase following it’s overexpression. In agreement with the observed loss of luminal Mn2+ we could observe concurrent reduction in many Mn2+-related process in the ER lumen. Conversely, cytosolic Mn2+-dependent processes were increased. Together, these data support a role for Spf1p in Mn2+ transport in the cell. We also demonstrate that the human sequence homologue, ATP13A1, is a functionally conserved orthologue. Since ATP13A1 is highly expressed in developing neuronal tissues and in the brain, this should help in the study of Mn2+-dependent neurological disorders

Glypicans as novel regulators of synaptic connectivity

The function of neural circuits depends on precise patterns of connectivity and the properties of synaptic transmission at specific synapses. The hippocampal mossy fiber (MF) synapse, which connects dentate gyrus granule cells with CA3 neurons, is a major information processing pathway and presents unique structural and functional properties. The molecular mechanisms governing MF synapse development and its specific structural-functional properties are largely unknown. Recently, we identified the presynaptic protein Glypican 4 (GPC4) as a novel synaptic organizing protein with a key role in excitatory synapse development. GPC4 is strongly enriched in the presynaptic compartment of MF synapses, but whether it is involved in MF synapse development or function is currently unknown. Using an in vivo knock down strategy we find that GPC4 controls the morphological maturation of MF presynaptic terminals. To dissect how GPC4 exerts this function, we performed a proteomic screen to identify novel GPC4 binding partners. We identified a largely uncharacterized, orphan G-protein coupled receptor, GPR158, as potential GPC4 interactor. Biochemical analysis demonstrates that GPC4 binds to GPR158 in a heparan sulfate-dependent manner, while localization studies indicate a specific GPR158 postsynaptic localization at the MF synapse. Further, we find that GPR158 stimulates presynaptic differentiation in cultured neurons in a GPC4-dependent manner. In GPR158 knockout mice, the characteristic presynaptic facilitation of MF synapses is strongly impaired. All together these data support the hypothesis that the GPC4-GPR158 interaction may act as MF synapse-specific interaction required for its development and function. We are currently dissecting the role of GPC4-GPR158 interaction in vivo during MF synapse development.status: publishe

Natura dei terremoti e comportamenti di sicurezza

I disastri sono eventi che hanno conseguenze potenzialmente negative per le persone, le cose e l’ambiente, comportando una sproporzione tra le richieste del contesto e le risorse degli individui atte a farvi fronte. Si distinguono principalmente in disastri naturali e disastri provocati dall’uomo o tecnologici. La comunicazione opportuna delle informazioni relative ai disastri è fondamentale per mitigarne gli effetti. La comunicazione del rischio può essere svolta in modo preventivo al fine di aumentare la preparazione individuale e la resilienza. A tale scopo, si possono implementare programmi di educazione ai disastri che, tuttavia, sono ancora rari. La percezione del rischio può essere influenzata da fattori individuali, familiari, relazionali e sociali. La scuola è un’arena ideale sia per strutturare la comunicazione relativa ai disastri sia per addestrare i giovani su come far fronte a un disastro prima che questo si verifichi, tenendo conto delle diverse caratteristiche in base alla fascia d’età. La resilienza permette di far fronte al rischio e agli eventi avversi e si manifesta tramite un adattamento psicologico positivo. Il progetto PrEmT permette di accrescere la resilienza in relazione al rischio sismico

Extracellular sphingosine-1-phosphate contributes to survival properties of glioblastoma stem cells

Extracellular sphingosine-1-phosphate contributes to survival properties of glioblastoma stem cells Riccitelli E., Giussani P., Di Vito C., Condomitti G., Tringali C., Galli R., Viani P., Riboni L. Department of Medical Biotechnology and Translational Medicine, University of Milan, LITA Segrate, Via Fratelli Cervi 93, 20090 Segrate, Milan, Italy ([email protected]) Sphingosine-1-phosphate (S1P) is a potent bioactive lipid formed from sphingosine by sphingosine kinases (SKs) action. S1P is considered as an onco-promoter molecule, favouring growth, invasion, and therapy-resistance of different tumours, including glioblastomas, the most frequent and aggressive human intracranial cancers. Despite the introduction of the alkylating agent temozolomide in glioblastoma therapy has improved patient survival, drug resistance limits its benefits. Accumulating literature indicates that glioblastoma stem-like cells (GSCs), a subpopulation of cells with the exclusive ability to self-renew and maintain the tumor, might contribute to glioblastoma aggressiveness and resistance to therapy. This study investigated S1P secretion by GSCs, and its possible role in cell survival. To this purpose we used GSCs isolated from the human U87 glioblastoma cell line (U-SC) and GSCs derived from a primary culture of human glioblastoma (L0627). We found that both GSC models efficiently form neurospheres in mitogen-defined medium, and express high levels of recognized neural-stem cell markers. Moreover, GSCs exhibited resistance to temozolomide, despite not expressing the DNA repair protein MGMT, a major contributor to temozolomide-resistance. Further analyses revealed the presence of S1P not only inside the cells, but also in the culture medium from both GSCs and U87. Notably the extracellular S1P level was found much higher in GSC models than in U87 cells, and the ratio between extracellular and intracellular S1P was 1:10 and 1:1 in U87 and GSCs, respectively. Enzyme activity assays excluded SKs presence in GSC medium, implicating an efficient secretion of S1P in GSCs. Intriguingly, concomitant treatment with temozolomide and a SKs inhibitor made GSCs sensitive to drug toxicity. Furthermore, S1P administration promoted cell survival after this co-treatment. Altogether our data implicate GSCs as an important source of extracellular S1P, which might act as an autocrine signal contributing to the survival properties of GSCs

Synapse type-specific proteomic dissection identifies IgSF8 as a hippocampal CA3 microcircuit organizer

Excitatory and inhibitory neurons are connected into microcircuits that generate circuit output. Central in the hippocampal CA3 microcircuit is the mossy fiber (MF) synapse, which provides powerful direct excitatory input and indirect feedforward inhibition to CA3 pyramidal neurons. Here, we dissect its cell-surface protein (CSP) composition to discover novel regulators of MF synaptic connectivity. Proteomic profiling of isolated MF synaptosomes uncovers a rich CSP composition, including many CSPs without synaptic function and several that are uncharacterized. Cell-surface interactome screening identifies IgSF8 as a neuronal receptor enriched in the MF pathway. Presynaptic Igsf8 deletion impairs MF synaptic architecture and robustly decreases the density of bouton filopodia that provide feedforward inhibition. Consequently, IgSF8 loss impairs excitation/inhibition balance and increases excitability of CA3 pyramidal neurons. Our results provide insight into the CSP landscape and interactome of a specific excitatory synapse and reveal IgSF8 as a critical regulator of CA3 microcircuit connectivity and function