1,721,014 research outputs found
Coimmunoprecipitation analysis of small conductance potassium channel type 3 assembly in hek-293 cell line
No full textA therapeutic perspective for proliferative vitreoretinopathy based on the inhibition of epithelial-mesenchymal transition by miR-194
No full textA Therapeutic Perspective for Proliferative Vitreoretinopathy Based on the Inhibition of Epithelial-Mesenchymal Transition by miR-19
Assembly and trafficking of human small conductance Ca2+ -activated K+ channel SK3 are governed by different molecular domains.
No full textIntracellular trafficking is an important event in the control of type and number of ion channels expressed on the cell surface. In this study, we have identified molecular domains involved in assembly and trafficking of the human small conductance Ca2+-activated K+ channel SK3. Deletion of the N-terminus, the C-terminus, or the calmodulin-binding domain (CaMBD) led to retention of SK3 channels in the endoplasmic reticulum. Presence of the CaMBD allowed trafficking to the Golgi complex, and sequences downstream were required for efficient transport to the plasma membrane, suggesting several steps in the control of SK3 forward trafficking. Co-immunoprecipitation studies demonstrated that SK3 subunits lacking the N-terminus, the CaMBD, or the distal C-terminus, but not the entire C-terminus, were able to oligomerize with wild-type SK3 subunits. Thus, these two C-terminal regions of SK3 seem to contribute to assembly and trafficking of channels whereas the N-terminus is necessary for trafficking but not sufficient for oligomerization
Homing and migration of transplanted leptomeningeal stem/progenitor cells in adult rat brain
No full textWe previously described stem/progenitor cells with neural differentiation potential in leptomeninges of postnatal day 15 (P15) rats. Leptomeninges, which include arachnoid and pia mater, cover the entire central nervous system (CNS) and are filled with cerebrospinal fluid (CSF) produced by choroid plexi. Long-term maintenance of stem cells requires their migration and homing within supportive stem cell niches. These processes are mediated by recognition processes with extravascular tissue-specific structures and interaction with chemotactic factors.
We found that leptomeninges of adult rats express important chemotactic factors, such as SDF-1, as well as several extracellular matrix components endowed of trophic functions, including laminin, fibronectin and agrin. Moreover, we found that nestin-positive cells in leptomeninges express CXCR4, the chemokine receptor specific for SDF-1.
To further characterize leptomeninges as a stem cell niche, we analysed the homing and the distribution in the brain of adult rats of transplanted and in vitro-expanded leptomeningeal stem/progenitor cells (LeSC).
Expanded leptomeningeal nestin-positive cells, derived from P15 EGFP-transgenic rats, were stereotaxically injected (3x103 EGFP+cells in 1μl) into the third ventriculus of adult rats (n=4) and observed by immunofluorescence confocal microscopy 4 weeks after transplantation. We found injected EGFP+/nestin+ cells in leptomeninges intermingled with resident nestin-positive cells.
EGFP+ cells were also observed in the cortex parenchima. Most of these cells were negative for both nestin and the neural markers MAP2, GFAP and NG2; some EGFP+/MAP2-positive cells were found. Interestingly, some of the EGFP+ cells were found along blood vessels, located in the perivascular space,in close contact with astrocytes, and the lumen of vessels.
At difference with intraventricular injection, in vitro-expanded EGFP+LeSC cells injected into the hippocampus expressed several neuronal markers including neurofilament, doublecortin, NeuN and Gad-67. Thus, adult neurogenic niches play a role in directing neuronal production.
In conclusion, in vitro expanded leptomeninges-derived nestin-positive cells injected in adult brain retained stem cell features when located in the leptomeninges and differentiated into neurons when located in the brain parenchyma, further supporting the LeSC plasticity. These data suggest leptomeninges are a favourable microenvironment, or niche, capable of hosting stem/precursor cells
Calcium imaging in living cells in cultures: a first-level-of-analysis approach for assessment of neuronal function
No full textBesides traditional methodologies for investigations on neuronal function, such as electrophysiology and neurochemistry, in-vivo living imaging has been recently used as a reliable method for first-level-of-analysis of the functionality of neural systems.
The aims of our research are focused on the expression of molecular markers of plasticity together with the evaluation of responses to different stimuli applications in neuronal primary culture. The experimental strategy is therefore based on set up in non-neuronal cells, and then in neuronal cell culture, calcium imaging and immunofluorescence assays, respectively applied in subsequent manner.
First step was the development of assays in a non-neuronal cell line (mouse pheochromocytoma cells, PC12). Undifferentiated PC12 cells kept for 4-5 days in growing medium, were placed on glass slide and differentiated with 50 ng/ml NGF-enriched medium for 5-6 days. Calcium imaging experiments were performed by applying 10, 25 or 55mM KCl on cells previously incubated with 5uM Fura2/AM. PC12 differentiated into neuron-like cells responded to 10, 25 or 55mM KCl in a dose-dependent manner.
Secondly, brain cortex was dissected from rat Sprague-Dawley at embryo stage E20, and then dissociated cells were cultured for 3 weeks in order to get primary neuronal cell cultures. Calcium imaging experiments were performed applying at different time-points 55 mM KCl or glutamatergic agonists, after incubation with 5μM Fura2/AM. Primary cultured cortical neurons responded to 55 mM KCl and to glutamatergic agonists.
On same cultures, immunofluorescence was then performed by using antibodies raised against microtubule associated protein (MAP2), glial fibrillary acid protein (GFAP), synaptophysine, postsynaptic marker (PSD-95) and glutamatergic receptors (GluR2, NR1). Immunofluorescence analysis by confocal microscopy highlighted glia cells positive for GFAP and neurons positive for the specific MAP2; moreover same neurons were also positive for synaptophysine, PSD95, and NR1, GluR2 glutamate receptors.
Calcium imaging assay was successfully set-up on PC12 cell line and effectively applied to rat primary neuronal cortical cultured cells. A combined protocol of calcium imaging and immunofluorescence would allow to have a concomitant evaluation of cortical neurons culture function and morphology, in order to correlate calcium related responses to expression of molecular markers of plasticity
Protocol to develop a preclinical severe contusive-compressive SCI mouse model for translational research
Full text linkComplete spinal cord injury (SCI) leads to irreversible neurological damage due to failed neural repair, with no effective therapies currently available. Here, we present a protocol to induce severe contusive-compressive SCI at thoracic T11 level in mouse using the NYU-MASCIS II impactor. We describe steps for performing laminectomy, inducing the injury, and validating it through functional and histological analysis. This protocol replicates key aspects of human secondary injury, making it valuable for preclinical testing of SCI therapies
Migration of meningeal cells in response to contusive spinal cord injury
No full textRecently, we have characterized adult brain and spinal cord meninges and demonstrated that meninges contain nestin-positive cells, endowed with self-renewal and proliferative properties, and neural precursor doublecortin (DCX)-positive cells (Bifari et la 2009, Decimo et al 2011). Cells extracted from meninges can be expanded as neurospheres and induced to specifically differentiate in vitro into either functional neurons or mature oligodendrocytes. Strikingly, following contusive spinal cord injury (SCI), meningeal stem/precursor cells proliferate and increase in number, suggesting that they are activated by injury.
In this study we ask whether activated menigneal stem/precursor cells migrate from meninges to parenchyma upon injury-induced activation. In order to trace the meningeal stem/precursor cells we transduced meningeal cells in vivo with a lentiviral GFP vector.We found that meningeal stem/precursor cells migrate to the lesion site, express neuronal markers and persist in this region until 1 month post injury. This results suggest that meningeal stem/precursor cells may have a role in the parenchymal regenerative potential following SCI
In vitro characterization of the differentiation of meningeal stem/precursor cells into the oligodendrocyte lineage
No full textIn most diseases of the spinal cord, demyelination plays an important role in the generation and progression of the neurodegenerative lesion. Despite the fact that the spinal cord shows potential for regeneration of Schwann cells, this is limited and apparently insufficient for repair. Attempts to establish regenerative approaches by neural stem cell transplantation have been so far limited by lack of knowledge of the function of resident SCs and of adequate sources of cells.
We have recently described that Leptomeninges of the spinal cord are a niche for neural stem cells (Decimo et al, 2011). These Leptomeningeal Stem Cells (LeSCs) are endowed with self-renewal properties and can differentiate into neurons and oligodendrocytes when stimulated with the appropriate factors.
Considering that remyelinization may be a realistic and efficient goal for transplantation-based regenerative therapies of the spinal cord, we decided to define conditions for efficient and high-yield generation of precursor and differentiated cells of the oligodendrocyte lineage. In conclusion, we have established an efficient and reproducible method to generate large numbers of cells of oligodendrocyte-lineage at different and controlled levels of differentiation. Testing of remyelinization efficiency by different sets of cells will be performed in vitro and by transplantation experiments in vivo using animal models of traumatic lesion of the spinal cord and of multiple sclerosis
Immunofluorescence analysis of small conductance potassium channel type 3 (SK3) trafficking in hippocampal cells
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