Patrizia Saladino

Oligodendroglioma cells shed microvesicles which contain TRAIL as well as molecular chaperones and induce cell death in astrocytes

Microvesicles (MVs) shed from G26/24 oligodendroglioma cells were previously reported to cause a reproducible, dose-dependent, inhibitory effect on neurite outgrowth, and eventually neuronal apoptosis, when added to primary cultures of rat cortical neurons. These effects were reduced but not abolished by functional monoclonal antibodies against Fas-L. In order to investigate whether MVs contain other factors able to induce cell death, we tested them for TRAIL and found clear evidence of its presence in the vesicles. This finding suggests the possibility that Fas-L and TRAIL cooperate in inducing brain cell death. Aimed at understanding the route through which the vesicles deliver their messages to the target cells, we labeled oligodendroglioma cells with radioactive methionine and then added the labeled vesicles shed from tumor cells to unlabeled astrocytes in culture. Here we report that labeled proteins were delivered to the test cells. In order to investigate whether astrocytes, like neurons, are sensitive to oligodendroglioma-derived vesicles, MVs were prepared from media conditioned by G26/24 oligodendroglioma cells and added to primary cultures of rat cortical astrocytes. These cells were clearly more resistant than neurons to microvesicle-induced damage: a high dose (40 µg) of shed MVs induced cell death in only about 40% of astrocytes. Finally, we demonstrated that Hsp70 is specifically enriched in MVs which also contain, even if at lower level, the Hsc70 constitutive chaperone.

Oligodendroglioma cells synthesize the differentiation-specific linker histone H1˚ and release it into the extracellular environment through shed vesicles

Chromatin remodelling can be involved in some of the epigenetic modifications found in tumor cells. One of the mechanisms at the basis of chromatin dynamics is likely to be synthesis and incorporation of replacement histone variants, such as the H1° linker histone. Regulation of the expression of this protein can thus be critical in tumorigenesis. In developing brain, H1° expression is mainly regulated at the post-transcriptional level and RNA-binding proteins (RBPs) are involved. In the past, attention mainly focused on the whole brain or isolated neurons and little information is available on H1° expression in other brain cells. Even less is known relating to tumor glial cells. In this study we report that, like in maturing brain and isolated neurons, H1° synthesis sharply increases in differentiating astrocytes growing in a serum-free medium, while the corresponding mRNA decreases. Unexpectedly, in tumor glial cells both H1° RNA and protein are highly expressed, in spite of the fact that H1° is considered a differentiation-specific histone variant. Persistence of H1° mRNA in oligodendroglioma cells is accompanied by high levels of H1° RNA-binding activities which seem to be present, at least in part, also in actively proliferating, but not in differentiating, astrocytes. Finally, we report that oligodendroglioma cells, but not astrocytes, release H1° protein into the culture medium by shedding extracellular vesicles. These findings suggest that deregulation of H1° histone expression can be linked to tumorigenesis.

Identification in the rat brain of a set of nuclear proteins interacting with H1° mRNA.

Synthesis of H1° histone, in the developing rat brain, is also regulated at post-transcriptional level. Regulation of RNA metabolism depends on a series of RNA-binding proteins (RBPs); therefore, we searched for H1° mRNA-interacting proteins. With this aim, we used in vitro transcribed, biotinylated H1° RNA as bait to isolate, by a chromatographic approach, proteins which interact with this mRNA, in the nuclei of brain cells. Abundant RBPs, such as heterogeneous nuclear ribonucleoprotein (hnRNP) K and hnRNP A1, and molecular chaperones (heat shock cognate 70, Hsc70) were identified by mass spectrometry. Western blot analysis also revealed the presence of cold shock domain-containing protein 2 (CSD-C2, also known as PIPPin), a brain-enriched RBP previously described in our laboratory. Co-immunoprecipitation assays were performed to investigate the possibility that identified proteins interact with each other and with other nuclear proteins. We found that hnRNP K interacts with both hnRNP A1 and Hsc70 whereas there is no interaction between hnRNP A1 and Hsc70. Moreover, CSD-C2 interacts with hnRNP A1, Y box-binding protein 1 (YB-1), and hnRNP K. We also have indications that CSD-C2 interacts with Hsc70. Overall, we have contributed to the molecular characterization of a ribonucleoprotein particle possibly controlling H1° histone expression in the brain.

RNA-binding activity of the rat calmodulin-binding PEP-19 protein and of the long PEP-19 isoform

Synthesis of H1˚ histone protein, in the developing rat brain, seems to be regulated mainly at the post-transcriptional level. Since regulation of RNA metabolism depends on a series of RNA-binding proteins, we have been searching for RNA-binding proteins involved in the post-transcriptional regulation of the H1˚ gene. We recently reported isolation, from a cDNA expression library, of an insert encoding a novel protein, the C-terminal half of which is identical to that of PEP-19, a brain-specific protein involved in calcium metabolism. The novel protein was called long PEP-19 isoform (LPI). Herein we show that LPI, as well as PEP-19, can bind H1˚ RNA. Moreover, in order to improve production of functional LPI/PEP-19, we modified the protocol normally adopted for preparing histidine tagged-proteins from bacteria, by adding an additional purification step. We also found that both LPI and PEP can compete for H1˚ RNA binding with PIPPin (CSD-C2), another RNA-binding protein previously discovered in our laboratory. Since PEP19/LPI contain a calmodulin binding domain, we finally investigated whether their ability to bind RNA is affected by calmodulin. Our results show that calmodulin interferes with binding of H1˚ RNA to both PEP-19 and LPI, while it is not able to bind RNA on its own. This finding suggests that calcium/calmodulin may have a role in controlling H1˚ mRNA metabolism in the developing brain.

PPARα gene variants as predicted performance-enhancing polymorphisms in professional Italian soccer players

Background The PPARα gene encodes the peroxisome proliferator-activator receptor alpha, a central regulator of expression of other genes involved in fatty acid metabolism. The purpose of this study was to determine the prevalence of G allele of the PPARα intron 7 G/C polymorphism (rs4253778) in professional Italian soccer players. Methods Sixty professional soccer players and 30 sedentary volunteers were enrolled in the study. Samples of venous blood were obtained at rest, in the morning, by conventional clinical procedures; blood serum was collected and total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, and triglycerides were measured. An aliquot of anticoagulant-treated blood was used to prepare genomic DNA from whole blood. The G/C polymorphic site in PPARα intron 7 was scanned by using the PCR-RFLP (polymerase chain reaction restriction fragment length polymorphism) protocol with TaqI enzyme. Results We found variations in genotype distribution of PPARα polymorphism between professional soccer players and sedentary volunteers. Particularly, G alleles and the GG genotype were significantly more frequent in soccer players compared with healthy controls (64% versus 48%). No significant correlations were found between lipid profile and genotype background. Conclusion Previous results demonstrated an association of intron 7 G allele as well as the GG genotype in endurance athletes. Our result suggests that this is the case also in professional soccer players.

Monitoring biomarkers during preseason preparation period in professional soccer players

Aim. The present study aimed at investigating the effect of a 3-week experimental intervention on biomarkers in professional soccer players during the preseason preparation-period. Methods. Eight participants (age 22.5±2.2 yrs) were enrolled in the study. During the physical preparation period players have attended a training program (51,9 hours) planned by coaches of “Equipe-Sicilia-2009”. Results. At rest, the lipid profile, the creatine kinase (CK), the lactic-acid dehydrogenase (LDH) and the expression of nuclear receptors peroxisome-proliferator-activated receptors (PPAR α/γ) were analyzed before starting and after 3 weeks of training sessions. The plasma level of CK in our samples showed great variability already in the baseline: value was on average nearly 500 IU/l showed that a large amount of these athletes were a high responders. The CK level decreased (p<0.01) after 3 weeks of training. No significant changes were found in the LDH plasma level, in the lipid profile and in the expression of mRNA of PPAR α/γ and also no significant person’s correlations were found among variables. Conclusion. We retain that those basal biomarkers, except CK, might not be an effective support for coaches to better understand training adaptations and overreaching mechanisms during a 3-week of preseason preparation-period. More studies are necessary to confirm these results.