Tunneling nanotubes and mesenchymal stem cells: new insights into the role of melatonin in neuronal recovery

none5sìEfficient cell-to-cell communication is essential for tissue development, homeostasis, and the maintenance of cellular functions after injury. Tunneling nanotubes (TNTs) have emerged as a new important method of cell-to-cell communication. TNTs are primarily established between stressed and unstressed cells and can transport a variety of cellular components. Mitochondria are important trafficked entities through TNTs. Transcellular mitochondria transfer permits the incorporation of healthy mitochondria into the endogenous network of recipient cells, changing the bioenergetic profile and other functional properties of the recipient and may allow the recipient cells to recuperate from apoptotic processes and return to a normal operating state. Mesenchymal cells (MSCs) can form TNTs and transfer mitochondria and other constituents to target cells. This occurs under both physiological and pathological conditions, leading to changes in cellular energy metabolism and functions. This review summarizes the newly-described capacity of melatonin to improve mitochondrial fusion/fission dynamics and promote TNT formation. This new evidence suggests that melatonin’s protective effects could be attributed to its ability to prevent mitochondrial damage in injured cells, reduce senescence, and promote anastasis, a natural cell recovery phenomenon that rescues cells from the brink of death. The modulation of these new routes of intercellular communication by melatonin could play a key role in increasing the therapeutic potential of MSCs.openLuchetti, Francesca; Carloni, Silvia; Nasoni, Maria G.; Reiter, Russel J.; Balduini, WalterLuchetti, Francesca; Carloni, Silvia; Nasoni, Maria G.; Reiter, Russel J.; Balduini, Walte

Mouse model of panic disorder: Vulnerability to early environmental instability is strain-dependent.

AbstractEarly life experiences and genetic background shape phenotypic variation. Several mouse models based on early treatments have evaluated short‐ and long‐term phenotypic alterations and explored their molecular mechanisms. The instability of maternal cues was used to model human separation anxiety in outbred mice, one of the etiopathogenetic factors that predict panic disorder (PD). Application of the repeated cross‐fostering (RCF) protocol to inbred strains (C57 and DBA) allowed us to measure differential responses to the same experimental manipulation. Ultrasounds emitted during isolation indicated that after RCF, pups from both strains lose their ability to be comforted by nest cues, but the frequency modulation of separation calls increased in RCF‐C57 and decreased in RCF‐DBA mice. No strain‐specific difference in olfactory ability explained these responses in RCF‐exposed mice. Rather, disruption of the infant‐mother bond may differentially affect separation calls in the two strains. Moreover, the RCF‐associated increased respiratory response to hypercapnia–an endophenotype of human PD documented among mice outbred strains–was replicated in the C57 strain only. We suggest that RCF‐induced instability of the early environment affects emotionality and respiratory physiology differentially, depending on pups' genetic background. These strain‐specific responses provide a lead to understand differential vulnerability to emotional disorders

Melatonin reshapes the mitochondrial network and promotes intercellular mitochondrial transfer via tunneling nanotubes after ischemic-like injury in hippocampal HT22 cells

Mitochondrial dysfunction is considered one of the hallmarks of ischemia/reperfusion injury. Mitochondria are plastic organelles that undergo continuous biogenesis, fusion, and fission. They can be transferred between cells through tunneling nanotubes (TNTs), dynamic structures that allow the exchange of proteins, soluble molecules, and organelles. Maintaining mitochondrial dynamics is crucial to cell function and survival. The present study aimed to assess the effects of melatonin on mitochondrial dynamics, TNT formation, and mitochondria transfer in HT22 cells exposed to oxygen/glucose deprivation followed by reoxygenation (OGD/R). The results showed that melatonin treatment during the reoxygenation phase reduced mitochondrial reactive oxygen species (ROS) production, improved cell viability, and increased the expression of PGC1α and SIRT3. Melatonin also preserved the expression of the membrane translocase proteins TOM20 and TIM23, and of the matrix protein HSP60, which are involved in mitochondrial biogenesis. Moreover, it promoted mitochondrial fusion and enhanced the expression of MFN2 and OPA1. Remarkably, melatonin also fostered mitochondrial transfer between injured HT22 cells through TNT connections. These results provide new insights into the effect of melatonin on mitochondrial network reshaping and cell survival. Fostering TNTs formation represents a novel mechanism mediating the protective effect of melatonin in ischemia/reperfusion injury

Melatonin Attenuates Ischemic-like Cell Injury by Promoting Autophagosome Maturation via the Sirt1/FoxO1/Rab7 Axis in Hippocampal HT22 Cells and in Organotypic Cultures

Dysfunctional autophagy is linked to neuronal damage in ischemia/reperfusion injury. The Ras-related protein 7 (Rab7), a member of the Rab family of small GTPases, appears crucial for the progression of the autophagic flux, and its activity is strictly interconnected with the histone deacetylase Silent information regulator 1 (Sirt1) and transcription factor Forkhead box class O1 (FoxO1). The present study assessed the neuroprotective role of melatonin in the modulation of the Sirt1/FoxO1/Rab7 axis in HT22 cells and organotypic hippocampal cultures exposed to oxygen-glucose deprivation followed by reoxygenation (OGD/R). The results showed that melatonin re-established physiological levels of autophagy and reduced propidium iodide-positive cells, speeding up autophagosome (AP) maturation and increasing lysosomal activity. Our study revealed that melatonin modulates autophagic pathways, increasing the expression of both Rab7 and FoxO1 and restoring the Sirt1 expression affected by OGD/R. In addition, the Sirt1 inhibitor EX-527 significantly reduced Rab7, Sirt1, and FoxO1 expression, as well as autolysosomes formation, and blocked the neuroprotective effect of melatonin. Overall, our findings provide, for the first time, new insights into the neuroprotective role of melatonin against ischemic injury through the activation of the Sirt1/FoxO1/Rab7 axis

Synthesis and biological characterization of a new fluorescent probe for vesicular trafficking based on polyazamacrocycle derivative

The fluorescent probes represent an important tool in the biological study, in fact characterization of cellular structures and organelles are an important tool-target for understanding the mechanisms regulating most biological processes. Recently, a series of polyamino-macrocycles based on 1,4,7,10-tetraazacyclododecane was synthesized, bearing one or two NBD units (AJ2NBD·4HCl) useful as sensors for metal cations and halides able to target and to detect apolar environment, as lipid membranes. In this paper, we firstly illustrate the chemical synthesis of the AJ2NBD probe, its electronic absorption spectra and its behavior regarding pH of the environment. Lack of any cellular toxicity and an efficient labelling on fresh, living cells was demonstrated, allowing the use of AJ2NBD in biological studies. In particular, this green fluorescent probe may represent a potential dye for the compartments involved in the endosomal/autophagic pathway. This research's field should benefit from the use of AJ2NBD as a vesicular tracer, however, to ensure the precise nature of vesicles/vacuoles traced by this new probe, other more specific tests are needed

LFA-1 antigen identifies immature stages of human NK cell differentiation

Background. Human Natural killer (NK) cells are characterized by NK cell receptors (NKRs) with inhibitory and activatory function that finely control their functional activities. In particular, they express inhibitory receptors for MHC class I molecules, named killer cell immunoglobulin (Ig)-like receptors (KIRs) and C-type lectin CD94-CD159a, and many triggering molecules like NKp30, NKp44, NKp46, (called natural cytotoxicity receptors, NCRs), NKG2D, CD161, and CD244. The majority of peripheral blood human NK cells are characterized by a phenotype with a low density expression of CD56 (CD56dim) and a high expression of CD16 (CD16bright), whereas a minority (approximately 5–10%) shows a bright expression of CD56 (CD56bright). This latter NK subset presents relatively high expression of some cytokine receptors (CD117 and CD25) and the CD94-CD159a heterodimeric inhibitory receptor. CD56bright NK cells are widely expressed in lymphoid tissues and can be generated from CD34+ cells when cultured with combinations of flt-3 ligand (FL) or stem cell factor plus IL-15 or IL-2. During their development, NK cells sequentially acquire many different antigens but there is still limited knowledge on differentiation antigens able to identify immature human NK cells and the specific sequence through which developing NK cells acquire the expression of NKR.Methods. NK cells obtained from human CD34+ hematopoietic progenitor cells after 30-day culture with FL plus IL-15, or from peripheral and umbilical cord blood samples were characterized.Results. Virtually, all CD56 NK cells differentiated in vitro expressed CD117, CD25, NCRs, NKG2D, CD161, and CD244, while only a subset expressed CD18-CD11a (LFA-1), and CD94 molecule, defining an immature CD56bright/NCRs+/NKG2D+/LFA-1-/CD94- subset. Another small subset of cells expressing CD94 but not LFA-1 integrin was also identified, suggesting that during NK differentiation LFA-1 might be upregulated later than CD94. To verify this hypothesis in vivo, we evaluated the NK cell expression of LFA-1 in both peripheral and umbilical cord blood samples. Interestingly, in these blood fluids, we have identified a lineage negative CD34-/LFA-1low/NKp46dim/NKG2Ddim/CD94- subset that resembled an immature stage of NK cells present in lymph nodes. Conclusions. Altogether, the results indicate that CD18-CD11a integrin, as well as CD11b in mice, may be a useful marker to identify immature stages of human NK cell differentiation

Rapamycin Re-Directs Lysosome Network, Stimulates ER-Remodeling, Involving Membrane CD317 and Affecting Exocytosis, in Campylobacter Jejuni-Lysate-Infected U937 Cells.

The Gram-negative Campylobacter jejuni is a major cause of foodborne gastroenteritis in humans worldwide. The cytotoxic effects of Campylobacter have been mainly ascribed to the actions of the cytolethal distending toxin (CDT): it is mandatory to put in evidence risk factors for sequela development, such as reactive arthritis (ReA) and Guillain-Barré syndrome (GBS). Several researches are directed to managing symptom severity and the possible onset of sequelae. We found for the first time that rapamycin (RM) is able to largely inhibit the action of C. jejuni lysate CDT in U937 cells, and to partially avoid the activation of specific sub-lethal effects. In fact, we observed that the ability of this drug to redirect lysosomal compartment, stimulate ER-remodeling (highlighted by ER-lysosome and ER-mitochondria contacts), protect mitochondria network, and downregulate CD317/tetherin, is an important component of membrane microdomains. In particular, lysosomes are involved in the process of the reduction of intoxication, until the final step of lysosome exocytosis. Our results indicate that rapamycin confers protection against C. jejuni bacterial lysate insults to myeloid cells

Role of melatonin in HT22 cells challenged with serum deprivation

In vitro serum deprivation (SD) is one model for investigating the molecular mechanisms underlying apoptosis as well as autophagy, which generally function as defense strategies upon cell injury by eliminating damaged organelles [1]. Furthermore, SD injury in vitro is widely used to mimic the ischemic environment [2]. In serum deprived conditions, cells show different parameters of apoptosis and autophagy. Melatonin (MLT), a lipophilic indole secreted by pineal and non-pineal cells, is a well-known potent free radical scavenger acting as neuroprotective molecule that prevents apoptotic cell death in several models of neurodegenerative diseases. In the present study we investigated the neuroprotective effects of MLT during SD condition on mouse hippocampal HT22 cells, considering that intracellular ROS are usually linked to autophagy and apoptosis. To explore potential effects of combining SD with melatonin we studied clonogenic survival of HT22 cells. Clonogenic assay demonstrated a significative (p< 0.01) reduction of HT22 total cell numbers challenged for 24h with SD, whereas the pre-treatment with 200nM of MLT for 24hr noticeably reduced this effect of about 30%. In HT22 starved cells the percentage of MitoTracker Red (MTR) positive cells doubled (P< 0.05) if compared to the control condition, suggesting that SD induced a remodelling of mitochondrial network. It is noteworthy that MLT pre-treatment produced a MTR positivity similar to that of controls. We next investigated whether melatonin was able to influence the autophagic pathway. Autophagy was detected by measuring the aggregation of LC3B protein coupled to green fluorescence protein (GFP). Confocal images show that SD induced an increase in the GFP-LC3 puncta, whereas the melatonin treatment reduces these aggregations. Taken together, our results suggest that MLT treatment may play protective roles against cellular modifications induced by SD treatment in HT22 cells

CD34 selected cells for the treatment of poor graft function after allogeneic stem cell transplantation.

Abstract Poor graft function (PGF) is characterized by pancytopenia and a hypoplastic marrow, with complete donor chimerism, usually without severe graft-versus-host disease (GVHD). We report 41 patients with PGF, treated with granulocyte colony-stimulating factor–mobilized CD34 selected cells, at a median interval from transplant of 140 days, without conditioning and without GVHD prophylaxis. Donors were HLA matched siblings (n = 12), unrelated donors (n = 18), or mismatched family members (n = 11). The median number of infused CD34 + cells was 3.4 × 10 6 /kg. The rate of trilineage recovery was 75%: 83% for HLA matched siblings and 72% for unrelated and mismatched family members ( P = .3). The cumulative incidence of acute grade II GVHD was 15%, and no patient developed de novo chronic GVHD. The actuarial 3-year survival is 63%: 76% and 25% for patients with or without trilineage recovery. These data confirm the role of CD34 + selected cells from the same donor in the treatment of PGF and warrant the request for a second donation also when the donor is unrelated