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IL TRASPORTO DI CLORURO: COINVOLGIMENTO IN ALCUNE PATOLOGIE UMANE
Full text linkSeveral human inherited diseases are caused by mutations in chloride channels or transporters, which cause symptoms as diverse as epilepsy, startle disease, deafness, blindness, lysosomal storage and neurodegeneration, osteopetrosis, lung infections and fibrosis, male infertility, renal salt loss, and kidney stones, clearly indicating the crucial importance of anion transport in many tissues. ICln is a water-soluble protein forming a PH domain, which can be introduced into the cellular membrane to form ion channels. When expressed in cellular systems, wt-ICln ion channels mediate a chloride current resembling those activated after cell swelling (ICl,swell), suggesting a role for ICln in cell volume regulation. During the first part of my PhD program at the department of Pharmacology and Toxicology (Paracelsus Medical University of Salzburg, Austria) I was involved in the functional characterization (by Black Lipid Bilayer and Patch Clamp experiments) of a new mutant form of the ICln protein possibly implicated in a heart disease. The object of my investigation was the insertion of a Thymine in position 383 in the nucleotide sequence. This mutation produces a frameshift, leading to a scrambled sequence of 12 aminoacids (starting from the aminoacid 128) and (an earlier termination (A128FSX139) of the channel sequence. This mutation has been identified in a patient whose familiarity, symptoms and QT value suggested she was suffering from long QT syndrome (LQTS).
Black lipid Bilayer experiments showed that the mutated protein reconstituted in artificial membrane of sphingomyelin had the same electrophysiological characteristics of the wild type protein. Otherwise Patch Clamp experiments (whole cell configuration) showed that the overexpression of the mutated protein in HEK293 Phoenix cells produced a significantly reduced ICl,swell current in comparison to that obtained by hIClnWT overexpression. During the second part of my PhD program I was involved in the functional characterization of the 5`flanking region of SLC26A4. SLC26A4 (Pendrin) was cloned by positional characterization of the gene for the Pendred syndrome (OMIM #274600), a recessively inherited disorder causing congenital deafness and thyroid goiter accountable for up to 10% of inherited hearing loss. The purpose of this part of my work was to identify the minimum sequence necessary for the transcription of the SLC26A4 gene and, when possible, to identify sequences important for the “tissue specific“ expression of the gene and for the recognition of responsive elements in a promoter sequence. The experiments performed in both HEK 293 Phoenix cells and in rat thyroid PC-CL3 cells showed that a sequence of only 286 base pairs is able to drive the basal expression of the pendrin gene in both cellular models. We assumed that the sequence contains all the cis-acting signals necessary for the activity of the basal transcription machinery. Moreover experiment performed in PC-CL3 cells allowed us identifying a tireoglobuline (TG) responsive element of only 205 base pairs in the promoter sequence of pendrin protein. Through acquiring an in-depth knowledge of the mechanisms involved in SLC26A4 gene regulation it would be possible to intervene on pendrin expression in case of an anomalous regulation in order to improve symptoms found in patients suffering from PS
Implementation of the stem cell properties of NG2+cells : focus on the epigenetic modulator VPA and the purinergic receptor GPR17
No full textImplementation of the stem cell properties of NG2-expressing neural precursor cells by purinergic signaling: an innovative reparative approach to neurodegeneration
No full textCells expressing the proteoglycan NG2 are multipotent neural progenitors with lineage plasticity able to primarily generate myelinating oligodendrocytes. They are also known as oligodendrocyte precursors cells (OPCs), are the only slowly proliferating cells in the adult brain, and react to injury. Under some circumstances, they can also generate neurons (Kondo & Raff, Science 289:1754, 2000); however, this first sensational paper showing that NG2 cells can be reprogrammed to neurons was followed by only few in vitro (Liu et al., J Neurosci 27:7339, 2007) and in vivo confirmations (Richardson et al, Neuron 70:661, 2011). The latter study suggests that NG2 cells lineage plasticity may be markedly widened by insults and epigenetic agents that profoundly change their reactivity and the surrounding local mileu. Previous studies from our laboratory have identified the purinergic receptor GPR17 as a new marker of early stages of NG2 cell differentiation, showing that GPR17 activation accelerates NG2 cells’ oligodendrocyte fate (Fumagalli et al., JBC 25:10593, 2011; Ceruti et al., Glia, 59:363, 2011; Boda et al., Glia 59:1958, 2011).
Based on these premises, aim of this work is to unveil the stem cell properties of NG2+ precursor cells and to induce their differentiation towards a neuronal lineage, also through the modulation of the GPR17 receptor.
Primary OPCs from the cerebral cortex of P2 rat pups were kept in culture for 6-8 days as mixed astrocytes-OPCs cultures and purified by vigorous shaking of flasks, and immunopanning selection. OPCs have been then cultured accordingly to two published protocols (Kondo & Raff, 2000; Liu et al., 2007) both able to unveil their stem cell properties. In either protocol, we have verified if and how the exposure to various pharmacological agents (including GPR17 receptor ligands) can modulate OPCs plasticity and their differentiation to neurons. In particular, we have utilized the non-selective GPR17 agonist UDP-glucose (10 μM) and antagonist cangrelor (10 μM), in parallel to the anticonvulsant agent valproic acid (VPA, 500 μM). VPA is known as an epigenetic modulator, which inhibits histone deacethylases (HDAC) and consequently induces transcription changes which favor neurogenesis (Yu et al., Neuropharm 56:473, 2009).
In both culturing conditions, we observed an increase in the percentage of cells expressing the neuronal marker β-tubulinIII (βtubIII) upon treatment with cangrelor and VPA with respect to control. Interestingly GPR17, which is normally expressed only by NG2+ cells, was surprisingly detected in a subset of βtubIII+ cells already under control differentiative condition, suggesting its potential involvement in neurogenesis. The appearance of this cell population was further incremented by the exposure to VPA and cangrelor, and slightly reduced by UDP-glucose, which likely promotes receptor down-regulation and drove NG2 cell specification towards a “classical” oligodendrocyte fate.
Taken together, our results suggest that antagonizing GPR17 functions can address the fate of NG2 cells towards the generation of new neurons, as observed with epigenetic modulators like VPA. We are currently testing whether a combination of the two agents may further enhance the neurogenic shift of NG2 cells, a paradigm that could be exploited in neuroregeneration in vivo
PURINERGIC SIGNALING AND NEUROGENESIS: MODULATION OF ADULT BRAIN SUBVENTRICULAR ZONE CELL FUNCTIONS AND PARENCHIMAL PROGENITORS MULTIPOTENCY.
Full text linkDespite previous beliefs, the generation of new neurons and new glia in the central nervous system (CNS) continues throughout life. Adult neurogenesis occurs in both classical neurogenic niches (e.g., the subventricular zone, SVZ, of the lateral ventricles) as well as in the entire brain's parenchyma, which is full of quiescent neural progenitors that are activated after injury. In particular, NG2-positive polydendrocytes, that usually differentiate to mature oligodendrocytes participating to re-myelination after injury, retain some multipotency and, under some conditions, can also generate neurons and astrocytes (Nishiyama et al., 2009).
Among various neurotransmitters and growth factors, extracellular nucleotides (ATP, UTP, their break-down products and sugar nucleotides), which are released at high amounts at the sites of CNS damage (Ulrich et al., 2012) are key actors in regulating reparative responses via purinergic P2 receptors (Abbracchio et al., 2009).
Previous studies from our laboratory have identified the purinergic receptor GPR17 as a new marker of early stages of NG2 cell differentiation, showing that GPR17 activation accelerates NG2 cells’ oligodendrocyte fate (Fumagalli et al., 2011; Ceruti et al., 2011; Boda et al., 2011). Interestingly, GPR17 is also one of the key genes expressed by human adult neural stem cells (Maisel et al., 2007), suggesting a possible role in cell fate determination.
Based on these premises, the aim of my PhD project was to investigate the role of purinergic signaling in regulating stem cell proprieties of adult brain subventricular zone and of NG2+ parenchymal progenitors.
In the first part of my PhD thesis, by using a conditional GLAST::CreERT2 Rosa YFP mouse model and an in vitro neurosphere assay, we have demonstrated that the P2Y receptor agonist ADPβS promotes the proliferation of SVZ neural progenitors and sustains their progression towards the generation of neuroblasts, either directly or through the activation of parenchymal astrocytes.
In the second part of my PhD, OPCs have been then cultured accordingly to two published protocols (Kondo & Raff, 2000; Liu et al., 2007) both able to unveil their stem cell properties. In both protocols, we observed an increase in the percentage of cells expressing the neuronal marker β-tubulinIII (βtubIII) upon treatment with the non-selective GPR17 antagonist Cangrelor and VPA with respect to control. Interestingly GPR17, which is normally expressed only by NG2+ cells, was surprisingly detected in a subset of βtubIII+ cells already under control differentiative condition, suggesting its potential involvement in neurogenesis. The appearance of this cell population was further incremented by the exposure to VPA and Cangrelor. Taken together, our results suggest that antagonizing GPR17 functions can address the fate of NG2 cells towards the generation of new neurons, as observed with epigenetic modulators like VPA.
In conclusion, our results strengthen the evidence that the purinergic system crucially regulates neuronal progenitors, either in a classical neurogenic niche or in the brain parenchyma. 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
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 textModulation 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
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
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