169,784 research outputs found
Modulation of proliferation and differentiative potential of adult brain subventricular zone cells by purinergic signaling in vitro and in vivo: contribution of reactive astrocytes
No full textThe subventricular zone (SVZ) of the lateral ventricles is one of the two neurogenic regions persisting in the adult brain. Here, GFAP+ precursors (Type B cells) give raise to transit-amplifying Mash1+ Type C cells, which eventually further differentiate to doublecortin+ neuroblasts (Type A cells). It is now emerging that brain injuries boost neurogenesis in the adult SVZ, also through action on surrounding parenchyma or niche cells. Nevertheless, very few newborn neurons survive and integrate in the damaged areas, suggesting a non-permissive environment. Thus, understanding the pro- or anti-neurogenic activity of the various molecules composing the extracellular milieu of the neurogenic niche would greatly help designing appropriate pharmacological approaches to promote neurogenesis while reducing inhibitory signals.
In pathological conditions, the concentrations of extracellular nucleotides (eNTs) raise several folds, and contribute to reactive astrogliosis [Abbracchio & Ceruti, PUSI 2:595, 2006]. Among their multiple functions in brain parenchima, astrocytes are also key components of the neurogenic niche, and regulate neural stem cells (NSC) proliferation and differentiation. Moreover, also eNTs could directly modulate SVZ cells, possibly through the G protein-coupled P2Y1 nucleotide receptor [Mishra et al., Development, 33:675, 2006; Grimm et al., J Cell Sci 122:2524, 2009], although very few data are available especially in vivo.
In the present study, we tested the ability of the stable P2Y1 agonist ADPβS to control adult NSC activities, with a focus on the possible effects exerted by reactive astrocytes. ADPβS administration in the lateral ventricle of adult mice caused both reactive astrogliosis in the brain parenchyma and activation of SVZ progenitors. Indeed, proliferation of GFAP+ NSCs increased, leading to a significant expansion of the population of transit-amplifying Mash1+ progenitors and doublecortin+ neuroblasts in the SVZ. Lineage tracing experiments further demonstrated that ADPβS promoted GLAST+ progenitor proliferation, and sustained their progression towards the generation of rapidly dividing progenitors. Data were fully confirmed in vitro by the neurosphere assay, where ADPβS stimulated the differentiation of undissociated NS towards GFAP+ astrocytes, and β-IIITub+ neurons. To test whether ADPβS was acting directly on NSCs only or whether reactive astrocytes were involved, we grew NS in the conditioned media derived from Control astrocytic cultures or from astrocytes cultured in presence of ADPβS. Both astrocyte-conditioned medium reduced the number and size of primary NS with respect to control neurosphere medium. Notably, a significant enhancement in SVZ progenitor proliferation was observed when SVZ cells, initially grown in the supernatant of astrocytes exposed to ADPβS, were then shifted to normal medium. This suggests that ADPβS stimulates the release of yet-to-be identified mediator(s) whose removal boosted proliferation of SVZ cells. Our preliminary results from an ELISA assay suggest that IL-10 could likely play a role in this effect.
Taken together our results strengthen evidence that purinergic system crucially regulates SVZ progenitors, both directly and through the involvement of reactive astrocytes. The pharmacological modulation of the purinergic system could therefore represent a promising and innovative approach to exploit the intrinsic ability of the adult brain to regenerate in acute and chronic neurodegenerative disorders
Purinergic signaling modulates adult neurogenesis in the subventricular zone: role of parenchymal astrocytes
No full textBackground and Purpose - The subventricular zone (SVZ) of the lateral ventricles is one of the two neurogenic regions persisting in the adult brain [1]. Evidence is accumulating that neurogenesis in the SVZ is boosted following trauma or ischemia, also through the interaction with surrounding parenchyma or niche cells. Astrocytes are key components of the neurogenic niche, and play a vital role in regulating neural stem cells (NSC) proliferation and differentiation. However, the exact molecular mechanisms by which astrocytes modulate NSC functions have not been identified. Besides significantly contributing to reactive astrogliosis, increasing evidence suggests that extracellular nucleotides play a role in controlling adult neurogenesis; these functions become prominent especially under pathological conditions where nucleotides concentrations raise several folds. From the few data published so far, a primary role for the P2Y1 G protein-coupled receptor subtype is clearly emerging in controlling the proliferation and differentiative potential of SVZ cells [2]. Therefore, we tested the ability of ADP-beta-S, a stable P2Y1 receptor agonist, to modulate stem cell properties in the adult brain in vitro and in vivo, with a particular focus on the possible modulatory effects exerted by reactive astrocytes
Methods and Results – In vitro results: neurospheres (NS; floating aggregates of SVZ precursor cells, maintaining the ability to proliferate and self-renew in culture) were obtained by mouse SVZ [3] and grown in the absence or presence of ADPβS. When cells derived from the dissociation of SVZ were plated in the presence of ADPβS, an increased number of NS was generated with respect to cultures grown under control conditions. Moreover, ADPβS stimulated the differentiation of undissociated NS towards GFAP+ astrocytes, and β-IIITub+ neurons. Interestingly, a significant enhancement in secondary NS generation was detected when SVZ cells were initially grown as primary NS in the supernatant of astrocytic culture exposed to ADP-beta-S, and then shifted to normal medium. This suggests that extracellular nucleotide stimulate the release of yet-to-be identified astrocytic mediator(s) whose removal from the culture medium boosted the self-renewal capability of SVZ cells. Preliminary results reveal that ADPβS influence the release from astrocytes of several pro- and anti-inflammatory cytokines that could likely play a role in this effect.
In vivo results: a 7-day long intracerebroventricular (i.c.v.) administration of 100 μM ADPβS stimulated reactive astrogliosis in the brain parenchima surrounding the SVZ, and induced a massive reaction of GFAP-expressing precursors and astrocytes in the SVZ, which became hypertrophic. Moreover, ADPβS promoted BrdU incorporation, indicating a proliferative effect. Confirming in vitro data, ADPβS administration induced also a significant expansion of the population of Mash1+ transit-amplifying cells and of doublecortin+ neuroblasts. By taking advantage of a conditional GLAST::CreERT2 Rosa YFP mouse model, we also demonstrated that ADPβS promoted the proliferation of GLAST-expressing progenitors in the neurogenic niche, and sustained their progression towards the generation of rapidly dividing transit-amplifying cells.
Conclusions - Taken together, our data suggest that nucleotides can be used to increase the pool of NSCs and their differentiation towards neuroblasts, either directly or through the activation of parenchymal astrocytes. This effect could be exploited to restore brain functions following acute and chronic neurodegenerative disorders, by stimulating the self-repair intrinsic ability of the brain.
References
[1] Doetsch F, Garcia-Verdugo JM, Alvarez-Buylla A (1997) Cellular composition and three-dimensional organization of the subventricular germinal zone in the adult mammalian brain. J Neurosci 17:5046-5061.
[2] Suyama S, Sunabori T, Kanki H, Sawamoto K , Gachet C, Koizumi S, Okano H (2012) Purinergic signaling promotes proliferation of adult mouse subventricular zone cells. J Neurosci 32:9238-9247.
[2] Johansson C.B. et al., 1999. Exp.Cell Res. 253:733-736
MODULATION OF THE PROLIFERATION AND DIFFERENTIATIVE POTENTIAL OF ADULT BRAIN SUBVENTRICULAR ZONE CELLS BY PURINERGIC SIGNALING IN VITRO AND IN VIVO : CONTRIBUTION OF REACTIVE ASTROCYTES
No full textPenetration of ceftazidime into bronchial secretions in critically ill patients
No full textFive adult patients admitted to ICU, with respiratory failure, required endotracheal intubation and respiratory support. They were treated with ceftazidime because of lower respiratory tract infections. All patients were given ceftazidime at 1, 2 and 3 g dosages in a randomized sequence and timed serum and bronchial secretion samples collected. Mean peak serum concentrations of 60.3, 148.7 and 224.8 mg/L were found in the three treatment groups and mean trough levels of 1.02, 1.85 and 1.63 mg/L respectively. Ceftazidime appeared rapidly in bronchial secretions reaching mean maximal concentrations of 2.2, 4.81 and 5.69 mg/L in the first sampling period (0-2 h). Serum and bronchial secretions AUCs have been calculated showing that both almost doubled their values between 1 and 2 g dosing, while between 2 and 3 g doses only a moderate and non-significant increase were found. On the basis of these results, the existence of a saturable transport mechanism for ceftazidime from serum to bronchial secretions can be postulated
Purines regulate adult brain subventricular zone cell functions: Contribution of reactive astrocytes
No full textBrain injuries modulate activation of neural stem cells (NSCs) in the adult brain. In pathological conditions, the concentrations of extracellular nucleotides (eNTs) raise several folds, contribute to reactive gliosis, and possibly directly affect subventricular zone (SVZ) cell functioning. Among eNTs and derived metabolites, the P2Y1 receptor agonist ADP strongly promotes astrogliosis and might also influence SVZ progenitor activity. Here, we tested the ability of the stable P2Y1 agonist adenosine 5'-O-(2-thiodiphosphate) (ADPβS) to control adult NSC functions both in vitro and in vivo, with a focus on the possible effects exerted by reactive astrocytes. In the absence of growth factors, ADPβS promoted proliferation and differentiation of SVZ progenitors. Moreover, ADPβS-activated astrocytes markedly changed the pattern of released cytokines and chemokines, and strongly modulated neurosphere-forming capacity of SVZ progenitors. Notably, a significant enhancement in proliferation was observed when SVZ cells, initially grown in the supernatant of astrocytes exposed to ADPβS, were shifted to normal medium. In vivo, ADPβS administration in the lateral ventricle of adult mice by osmotic minipumps caused diffused reactive astrogliosis, and a strong response of SVZ progenitors. Indeed, proliferation of glial fibrillary acidic protein-positive NSCs increased and led to a significant expansion of SVZ transit-amplifying progenitors and neuroblasts. Lineage tracing experiments performed in the GLAST::CreERT2;Rosa-YFP transgenic mice further demonstrated that ADPβS promoted proliferation of glutamate/aspartate transporter-positive progenitors and sustained their progression toward the generation of rapidly dividing progenitors. Altogether, our results show that the purinergic system crucially affects SVZ progenitor activities both directly and through the involvement of reactive astrocytes
Salvemini Prof. Gaetano, sovversivo
No full textL'esilio di Getano Salvemini: l'uomo, lo storico, il fuoruscito che ha combattuto il fascismo per oltre vent'anni nell'analisi di alcuni fra i principali storici contemporanei dell'antifascismo, con filmati e immagini dell'epoca
Forming of ICln-dimers by disulphide bridging
No full textICln is a cytoplasmic water-soluble protein forming a PH domain, which can be intro-duced into the cellular membrane, and form ion channels. We set out to test, whether or not disulphide bridging and consecutive dimerization is important for channel formation. Wt-ICln ion channels when expressed in cellular systems, mediate a chloride current resembling that activated after cell swelling (IClswell ), which allows the cells to perform regulatory volume decrease (RVD). Knocking-down of ICln impairs IClswell, therefore supporting the hypothesis of ICln being an important entity for IClswell. In order to test the possible involvement of disulfide bonds in ICln dimerization, we examined the effect of the reducing agent dithiothreitol (DTT) on ICln dimerization using Western-blots we show that the ICln dimer is absent in samples treated with DTT. ICln has only two cystines (C128 and C130) located at the c-terminal end of the α-helix. To determine the specificity of DTT on ICln dimerization a double and single cystine mutant (C128A/C130A, C128A and C130A) were constructed and their effect on ICln dimeriza-tion under renaturing or non-denaturing conditions was analysed. Western-blots show that the C128A/C130A mutant annihilates the dimer formation. In addition to Western-blots, we performed a biophysical characterization of the mutants after their reconstitu-tion in black lipid bilayers built of a sphingomyeline mixture. By comparing ICln-wt and the different cystine mutants we found a dramatic decrease of channel formation, if disulphide bridging is impeded. Our experiments indicate that the format of disulphide bridging is essential for ICln function as a channel
Adult neurogenesis in the subventricular zone: studies on the role of purinergic signalling by the neurosphere assay
No full textAdult neurogenesis occurs in the subventricular zone (SVZ) and in the dentate gyrus of the hippocampus. Once isolated, cells from the SVZ can grow in vitro as clonal clusters of proliferating cells, called neurospheres (NS), which can either self-renew or differentiate to astrocytes, neurons and oligodendrocytes1. There is increasing evidence that nucleotides can play a role in controlling both adult and embryonic neurogenesis2. The aim of our study was to investigate the role of purinergic signaling in controlling the proliferation, the self-renewal capability and the differentiative potential of neural stem cells derived from adult murine SVZ by the NS assay.
To this aim, we evaluated if and how the P2Y1,11,12,13 non-selective agonist ADPβS can affect the proliferation and the self-renewal capability of NS. When analyzing the ability of cells deriving from primary NS to constitute new NS, we observed that ADPβS application increased the number of secondary NS, while it had no effect on the total number of viable cells and on the NS size. When the cells derived from the dissociation of secondary NS were used, ADPβS increased the number of tertiary NS whereas a reduction of the total number of viable cells and of the size of the NS was obtained. Since progenitor cells have limited proliferative and self-renewal capacity, and give rise to small-sized neurospheres3, it can be envisaged that ADPβS acts on this specific cell population and promotes the formation of a higher number of NS with a smaller size.
We also tested if the exposure to ADPβS changed the differentiative potential of 7-day-old dissociated primary NS, cultured as adherent cells for 7 additional days in presence of 2% FBS. No differences in the proportion among the various cell types derived from neurospheres between treated and untreated cultures were observed: the majority of the cells resulted to be GFAP-positive astrocytes, while few βIII-tubulin-positive neurons and O4-positive oligodendrocytes were detected. We are now evaluating the effect of ADPβS on the differentiative potential of 7-days-old secondary NS plated without dissociation and grown for 7 additional days without FBS and GFs. Preliminary results obtained by a densitometric analysis, suggest an increased presence of both βIII-tubulin+ neuron and GFAP+ astrocytes in ADPβS-treated cultures.
References
1. Doetsch F, Caille I, Lim DA, Garcia-Verdugo JM, Alvarez-Buylla A (1999) Subventricular zone astrocytes are neural stem cells in the adult mammalian brain. Cell, 97:703-716.
2. Neary JT and Zimmermann H (2009) Trophic functions of nucleotides in the central nervous system. Trends Neurosci, 32(4):189-98.
3. Gritti A, Bonfanti L, Doetsch F, Caille I, Alvarez-Buylla A, Lim DA, Galli R, Verdugo JM, Herrera DG, Vescovi AL (2002) Multipotent neural stem cells reside into the rostral extension and olfactory bulb of adult rodents. J Neurosci, 22(2):437-45
Tissue tropism and metabolic pathways of Midichloria mitochondrii suggest tissue-specific functions in the symbiosis with Ixodes ricinus.
No full textA wide range of arthropod species harbour bacterial endosymbionts in various tissues, many of them playing important roles in the fitness and biology of their hosts. In several cases, many different symbionts have been reported to coexist simultaneously within the same host and synergistic or antagonistic interactions can occur between them. While the associations with endosymbiotic bacteria have been widely studied in many insect species, in ticks such interactions are less investigated. The females and immatures of Ixodes ricinus (Ixodidae), the most common hard tick in Europe, harbour the intracellular endosymbiont “Candidatus Midichloria mitochondrii” with a prevalence up to 100%, suggesting a mutualistic relationship. Considering that the tissue distribution of a symbiont might be indicative of its functional role in the physiology of the host, we investigated M. mitochondrii specific localization pattern and the corresponding abundance in selected organs of I. ricinus females. We paired these experiments with in silico analysis of the metabolic pathways of M. mitochondrii, inferred from the available genome sequence, and additionally compared the presence of these pathways in seven other symbionts commonly harboured by ticks to try to obtain a comparative understanding of their biological effects on the tick hosts. M. mitochondrii was found to be abundant in ovaries and tracheae of unfed I. ricinus, and in ovaries, Malpighian tubules and salivary glands of semi-engorged females. These results, together with the in silico metabolic reconstruction allow to hypothesize that the bacterium could play multiple tissue-specific roles in the host, both enhancing the host fitness (supplying essential nutrients, enhancing the reproductive fitness, helping in the anti-oxidative defence, in the energy production and in the maintenance of homeostasis and water balance) and/or for ensuring its presence in the host population (nutrients acquisition, vertical and horizontal transmission). The ability of M. mitochondrii to colonize different tissues allows to speculate that distinctive sub-populations may display different specializations in accordance with tissue tropism. Our hypotheses should be corroborated with future nutritional and physiological experiments for a better understanding of the mechanisms underlying this symbiotic interaction
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