The unconventional secretion of stress-inducible protein 1 by a heterogeneous population of extracellular vesicles

The co-chaperone stress-inducible protein 1 (STI1) is released by astrocytes, and has important neurotrophic properties upon binding to prion protein (PrP^C). However, STI1 lacks a signal peptide and pharmacological approaches pointed that it does not follow a classical secretion mechanism. Ultracentrifugation, size exclusion chromatography, electron microscopy, vesicle labeling, and particle tracking analysis were used to identify three major types of extracellular vesicles (EVs) released from astrocytes with sizes ranging from 20–50, 100–200, and 300–400 nm. These EVs carry STI1 and present many exosomal markers, even though only a subpopulation had the typical exosomal morphology. The only protein, from those evaluated here, present exclusively in vesicles that have exosomal morphology was PrP^C. STI1 partially co-localized with Rab5 and Rab7 in endosomal compartments, and a dominant-negative for vacuolar protein sorting 4A (VPS4A), required for formation of multivesicular bodies (MVBs), impaired EV and STI1 release. Flow cytometry and PK digestion demonstrated that STI1 localized to the outer leaflet of EVs, and its association with EVs greatly increased STI1 activity upon PrP^C-dependent neuronal signaling. These results indicate that astrocytes secrete a diverse population of EVs derived from MVBs that contain STI1 and suggest that the interaction between EVs and neuronal surface components enhances STI1–PrP^C signaling.     

RIAM (Rap1-interacting adaptor molecule) regulates complement-dependent phagocytosis

Phagocytosis mediated by the complement receptor CR3 (also known as integrin α_Mß₂ or Mac-1) is regulated by the recruitment of talin to the cytoplasmic tail of the ß₂ integrin subunit. Talin recruitment to this integrin is dependent on Rap1 activation. However, the mechanism by which Rap1 regulates this event and CR3-dependent phagocytosis remains largely unknown. In the present work, we examined the role of the Rap1 effector RIAM, a talin-binding protein, in the regulation of complement-mediated phagocytosis. Using the human myeloid cell lines HL-60 and THP-1, we determined that knockdown of RIAM impaired α_Mß₂ integrin affinity changes induced by stimuli fMLP and LPS. Phagocytosis of complement-opsonized RBC particles, but not of IgG-opsonized RBC particles, was impaired in RIAM knockdown cells. Rap1 activation via EPAC induced by 8-pCPT-2′-O-Me-cAMP resulted in an increase of complement-mediated phagocytosis that was abrogated by knockdown of RIAM in HL-60 and THP-1 cell lines and in macrophages derived from primary monocytes. Furthermore, recruitment of talin to ß₂ integrin during complement-mediated phagocytosis was reduced in RIAM knockdown cells. These results indicate that RIAM is a critical component of the phagocytosis machinery downstream of Rap1 and mediates its function by recruiting talin to the phagocytic complement receptors.     

The LRRK2 G2019S mutant exacerbates basal autophagy through activation of the MEK/ERK pathway

Mutations in leucine-rich repeat kinase 2 (LRRK2) are a major cause of familial Parkinsonism, and the G2019S mutation of LRRK2 is one of the most prevalent mutations. The deregulation of autophagic processes in nerve cells is thought to be a possible cause of Parkinson’s disease (PD). In this study, we observed that G2019S mutant fibroblasts exhibited higher autophagic activity levels than control fibroblasts. Elevated levels of autophagic activity can trigger cell death, and in our study, G2019S mutant cells exhibited increased apoptosis hallmarks compared to control cells. LRRK2 is able to induce the phosphorylation of MAPK/ERK kinases (MEK). The use of 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene (U0126), a highly selective inhibitor of MEK1/2, reduced the enhanced autophagy and sensibility observed in G2019S LRRK2 mutation cells. These data suggest that the G2019S mutation induces autophagy via MEK/ERK pathway and that the inhibition of this exacerbated autophagy reduces the sensitivity observed in G2019S mutant cells.     

Extensive characterization of sphere models established from colorectal cancer cell lines

Links between cancer and stem cells have been proposed for many years. As the cancer stem cell (CSC) theory became widely studied, new methods were developed to culture and expand cancer cells with conserved determinants of “stemness”. These cells show increased ability to grow in suspension as spheres in serum-free medium supplemented with growth factors and chemicals. The physiological relevance of this phenomenon in established cancer cell lines remains unclear. Cell lines have traditionally been used to explore tumor biology and serve as preclinical models for the screening of potential therapeutic agents. Here, we grew cell-forming spheres (CFS) from 25 established colorectal cancer cell lines. The molecular and cellular characteristics of CFS were compared to the bulk of tumor cells. CFS could be isolated from 72 % of the cell lines. Both CFS and their parental CRC cell lines were highly tumorigenic. Compared to their parental cells, they showed similar expression of putative CSC markers. The ability of CRC cells to grow as CFS was greatly enhanced by prior treatment with 5-fluorouracil. At the molecular level, CFS and parental CRC cells showed identical gene mutations and very similar genomic profiles, although microarray analysis revealed changes in CFS gene expression that were independent of DNA copy-number. We identified a CFS gene expression signature common to CFS from all CRC cell lines, which was predictive of disease relapse in CRC patients. In conclusion, CFS models derived from CRC cell lines possess interesting phenotypic features that may have clinical relevance for drug resistance and disease relapse.     

Connexin 43 a check-point component of cell proliferation implicated in a wide range of human testis diseases

Gap junction channels link cytoplasms of adjacent cells. Connexins, their constitutive proteins, are essential in cell homeostasis and are implicated in numerous physiological processes. Spermatogenesis is a sophisticated model of germ cell proliferation, differentiation, survival, and apoptosis, in which a connexin isotype, connexin 43, plays a crucial role as evidenced by genomic approaches based on gene deletion. The balance between cell proliferation/differentiation/apoptosis is a prerequisite for maintaining levels of spermatozoa essential for fertility and for limiting anarchic cell proliferation, a major risk of testis tumor. The present review highlights the emerging role of connexins in testis pathogenesis, focusing specifically on two intimately interconnected human testicular diseases (azoospermia with impaired spermatogenesis and testicular germ cell tumors), whose incidence increased during the last decades. This work proposes connexin 43 as a potential cancer diagnostic and prognostic marker, as well as a promising therapeutic target for testicular diseases.     

The opportunistic pathogen Pseudomonas aeruginosa activates the DNA double-strand break signaling and repair pathway in infected cells

Highly hazardous DNA double-strand breaks can be induced in eukaryotic cells by a number of agents including pathogenic bacterial strains. We have investigated the genotoxic potential of Pseudomonas aeruginosa, an opportunistic pathogen causing devastating nosocomial infections in cystic fibrosis or immunocompromised patients. Our data revealed that infection of immune or epithelial cells by P. aeruginosa triggered DNA strand breaks and phosphorylation of histone H2AX (γH2AX), a marker of DNA double-strand breaks. Moreover, it induced formation of discrete nuclear repair foci similar to gamma-irradiation-induced foci, and containing γH2AX and 53BP1, an adaptor protein mediating the DNA-damage response pathway. Gene deletion, mutagenesis, and complementation in P. aeruginosa identified ExoS bacterial toxin as the major factor involved in γH2AX induction. Chemical inhibition of several kinases known to phosphorylate H2AX demonstrated that Ataxia Telangiectasia Mutated (ATM) was the principal kinase in P. aeruginosa-induced H2AX phosphorylation. Finally, infection led to ATM kinase activation by an auto-phosphorylation mechanism. Together, these data show for the first time that infection by P. aeruginosa activates the DNA double-strand break repair machinery of the host cells. This novel information sheds new light on the consequences of P. aeruginosa infection in mammalian cells. As pathogenic Escherichia coli or carcinogenic Helicobacter pylori can alter genome integrity through DNA double-strand breaks, leading to chromosomal instability and eventually cancer, our findings highlight possible new routes for further investigations of P. aeruginosa in cancer biology and they identify ATM as a potential target molecule for drug design.     

Regulation of oligodendrocyte precursor migration during development, in adulthood and in pathology

Oligodendrocytes are the myelin-forming cells in the central nervous system (CNS). These cells originate from oligodendrocyte precursor cells (OPCs) during development, and they migrate extensively from oligodendrogliogenic niches along the neural tube to colonise the entire CNS. Like many other such events, this migratory process is precisely regulated by a battery of positional and signalling cues that act via their corresponding receptors and that are expressed dynamically by OPCs. Here, we will review the cellular and molecular basis of this important event during embryonic and postnatal development, and we will discuss the relevance of the substantial number of OPCs existing in the adult CNS. Similarly, we will consider the behaviour of OPCs in normal and pathological conditions, especially in animal models of demyelination and of the demyelinating disease, multiple sclerosis. The spontaneous remyelination observed after damage in demyelinating pathologies has a limited effect. Understanding the cellular and molecular mechanisms underlying the biology of OPCs, particularly adult OPCs, should help in the design of neuroregenerative strategies to combat multiple sclerosis and other demyelinating diseases.     

Cell-penetrating peptide secures an efficient endosomal escape of an intact cargo upon a brief photo-induction

Since their discovery, cell-penetrating peptides (CPPs) have provided a novel, efficient, and non-invasive mode of transport for various (bioactive) cargos into cells. Despite the ever-growing number of successful implications of the CPP-mediated delivery, issues concerning their intracellular trafficking, significant targeting to degradative organelles, and limited endosomal escape are still hindering their widespread use. To overcome these obstacles, we have utilized a potent photo-induction technique with a fluorescently labeled protein cargo attached to an efficient CPP, TP10. In this study we have determined some key requirements behind this induced escape (e.g., dependence on peptide-to-cargo ratio, time and cargo), and have semi-quantitatively assessed the characteristics of the endosomes that become leaky upon this treatment. Furthermore, we provide evidence that the photo-released cargo remains intact and functional. Altogether, we can conclude that the photo-induced endosomes are specific large complexes-condensed non-acidic vesicles, where the released cargo remains in its native intact form. The latter was confirmed with tubulin as the cargo, which upon photo-induction was incorporated into microtubules. Because of this, we propose that combining the CPP-mediated delivery with photo-activation technique could provide a simple method for overcoming major limitations faced today and serve as a basis for enhanced delivery efficiency and a subsequent elevated cellular response of different bioactive cargo molecules.     

Overactive bone morphogenetic protein signaling in heterotopic ossification and Duchenne muscular dystrophy

Bone morphogenetic proteins (BMPs) are important extracellular cytokines that play critical roles in embryogenesis and tissue homeostasis. BMPs signal via transmembrane type I and type II serine/threonine kinase receptors and intracellular Smad effector proteins. BMP signaling is precisely regulated and perturbation of BMP signaling is connected to multiple diseases, including musculoskeletal diseases. In this review, we will summarize the recent progress in elucidation of BMP signal transduction, how overactive BMP signaling is involved in the pathogenesis of heterotopic ossification and Duchenne muscular dystrophy, and discuss possible therapeutic strategies for treatment of these diseases.