Drosophila Mpv17 forms an ion channel and regulates energy metabolism.

Mutations in MPV17 are a major contributor to mitochondrial DNA (mtDNA) depletion syndromes, a group of inherited genetic conditions due to mtDNA instability. To investigate the role of MPV17 in mtDNA maintenance, we generated and characterized a Drosophila melanogaster Mpv17 (dMpv17) KO model showing that the absence of dMpv17 caused profound mtDNA depletion in the fat body but not in other tissues, increased glycolytic flux and reduced lifespan in starvation. Accordingly, the expression of key genes of glycogenolysis and glycolysis was upregulated in dMpv17 KO flies. In addition, we demonstrated that dMpv17 formed a channel in planar lipid bilayers at physiological ionic conditions, and its electrophysiological hallmarks were affected by pathological mutations. Importantly, the reconstituted channel translocated uridine but not orotate across the membrane. Our results indicate that dMpv17 forms a channel involved in translocation of key metabolites and highlight the importance of dMpv17 in energy homeostasis and mitochondrial function.

Extracellular MicroRNA Signature of Human Helper T Cell Subsets in Health and Autoimmunity.

Upon T cell receptor stimulation, CD4+ T helper (Th) lymphocytes release extracellular vesicles (EVs) containing microRNAs. However, no data are available on whether human CD4+ T cell subsets release EVs containing different pattern of microRNAs. The present work aimed at filling this gap by assessing the microRNA content in EVs released upon in vitro T cell receptor stimulation of Th1, Th17, and T regulatory (Treg) cells. Our results indicate that EVs released by Treg cells are significantly different compared with those released by the other subsets. In particular, miR-146a-5p, miR-150-5p, and miR-21-5p are enriched, whereas miR-106a-5p, miR-155-5p, and miR-19a-3p are depleted in Treg-derived EVs. The in vitro identified EV-associated microRNA signature was increased in serum of autoimmune patients with psoriasis and returned to healthy levels upon effective treatment with etanercept, a biological drug targeting the TNF pathway and suppressing inflammation. Moreover, Gene Set Enrichment Analysis showed an over-representation of genes relevant for T cell activation, such as CD40L, IRAK1, IRAK2, STAT1, and c-Myb in the list of validated targets of Treg-derived EV miRNAs. At functional level, Treg-derived (but not Th1/Th17-derived) EVs inhibited CD4+ T cell proliferation and suppressed two relevant targets of miR-146a-5p: STAT1 and IRAK2. In conclusion, our work identified the miRNAs specifically released by different human CD4+ T cell subsets and started to unveil the potential use of their quantity in human serum to mark the pathological elicitation of these cells in vivo and their biological effect in cell to cell communication during the adaptive immune response.

Proteome profiling of neuroblastoma-derived exosomes reveal the expression of proteins potentially involved in tumor progression.

Neuroblastoma (NB) is the most common extracranial solid tumor in childhood, with grim prognosis in a half of patients. Exosomes are nanometer-sized membrane vesicles derived from the multivesicular bodies (MVBs) of the endocytic pathway and released by normal and neoplastic cells. Tumor-derived exosomes have been shown in different model systems to carry molecules that promote cancer growth and dissemination. In this respect, we have here performed the first characterization and proteomic analysis of exosomes isolated from human NB cell lines by filtration and ultracentrifugation. Electron microscopy demonstrated that NB-derived exosomes exhibited the characteristic cup-shaped morphology. Dynamic light scattering studies showed a bell-shaped curve and a polydispersity factor consistent with those of exosomes. Zeta potential values suggested a good nanoparticle stability. We performed proteomic analysis of NB-derived exosomes by two dimension liquid chromatography separation and mass spectrometry analyses using the multidimensional protein identification technology strategy. We found that the large majority of the proteins identified in NB derived exosomes are present in Exocarta database including tetraspanins, fibronectin, heat shock proteins, MVB proteins, cytoskeleton-related proteins, prominin-1 (CD133), basigin (CD147) and B7-H3 (CD276). Expression of the CD9, CD63 and CD81 tetraspanins, fibronectin, CD133, CD147 and CD276 was validated by flow cytometry. Noteworthy, flow cytometric analysis showed that NB-derived exosomes expressed the GD2 disialoganglioside, the most specific marker of NB. In conclusion, this study shows that NB-derived exosomes express a discrete set of molecules involved in defense response, cell differentiation, cell proliferation and regulation of other important biological process. Thus, NB-derived exosomes may play an important role in the modulation of tumor microenvironment and represent potential tumor biomarkers.

Liposome-mediated therapy of neuroblastoma.

Neuroblastoma (NB) is the most common extracranial solid tumor in childhood and the most frequently diagnosed neoplasm during infancy. Despite of aggressive treatment strategies, the 5-year survival rate for metastatic disease is still less than 60% and, consequently, novel therapeutic approaches are needed. For increasing the therapeutic index of anticancer drugs, while reducing side effects, one of the most promising strategies in modern chemotherapy is based on the development of innovative drug delivery systems, such as liposomes. "Anticancer drug"-loaded liposomes have demonstrated enhanced ability to target to the affected area, as well as increased antitumor efficacy compared to conventional drugs. Liposomes tend to extravasate preferentially and to accumulate into tumor interstitial fluids, due to the defective structure of the new angiogenic vessels within the tumor masses. This inherent tumor selectivity can be increased further by coupling tumor-specific antibodies or other targeting moieties to the surface of the lipid envelope. Here, we describe the methodology used in these studies, as well as the antitumor results obtained by the use of several "anticancer drugs," encapsulated into antibody- and peptide-targeted liposomal formulations, against NB.