P2X7 receptor activation awakes a dormant stem cell niche in the adult spinal cord.

The ependyma of the spinal cord is a latent stem cell niche that is reactivated by injury, generating new cells that migrate to the lesion site to limit the damage. The mechanisms by which ependymal cells are reactivated after injury remain poorly understood. ATP has been proposed to act as a diffusible "danger signal" to alert about damage and start repair. Indeed, spinal cord injury (SCI) generates an increase in extracellular ATP around the lesion epicenter that lasts for several hours and affects the functional outcome after the damage. The P2X7 receptor (P2X7r) has functional properties (e.g., low sensitivity for ATP, high permeability for Ca2+) that makes it a suitable candidate to act as a detector of tissue damage. Because ependymal cells express functional P2X7r that generate an inward current and regenerative Ca2+ waves, we hypothesize that the P2X7r has a main role in the mechanisms by which progenitor-like cells in the ependyma react to tissue damage. To test this possibility, we simulated the P2X7r activation that occurs after SCI by in vivo intraspinal injection of the selective agonist BzATP nearby the central canal. We found that BzATP rescued ependymal cells from quiescence by triggering a proliferative response similar to that generated by injury. In addition, P2X7r activation by BzATP induced a shift of ependymal cells to a glial fibrillary acidic protein (GFAP) phenotype similar to that induced by injury. However, P2X7r activation did not trigger the migration of ependyma-derived cells as occurs after tissue damage. Injection of BzATP induced the expression of connexin 26 (Cx26) in ependymal cells, an event needed for the proliferative reaction after injury. BzATP did not induce these changes in ependymal cells of P2X7-/- mice supporting a specific action on P2X7r. In vivo blockade of P2X7r with the potent antagonist AZ10606120 reduced significantly the injury-induced proliferation of ependymal cells. Our data indicate that P2X7r has a key role in the "awakening" of the ependymal stem cell niche after injury and suggest purinergic signaling is an interesting target to improve the contribution of endogenous progenitors to repair.

Is IIIG9 a New Protein with Exclusive Ciliary Function? Analysis of Its Potential Role in Cancer and Other Pathologies.

The identification of new proteins that regulate the function of one of the main cellular phosphatases, protein phosphatase 1 (PP1), is essential to find possible pharmacological targets to alter phosphatase function in various cellular processes, including the initiation and development of multiple diseases. IIIG9 is a regulatory subunit of PP1 initially identified in highly polarized ciliated cells. In addition to its ciliary location in ependymal cells, we recently showed that IIIG9 has extraciliary functions that regulate the integrity of adherens junctions. In this review, we perform a detailed analysis of the expression, localization, and function of IIIG9 in adult and developing normal brains. In addition, we provide a 3D model of IIIG9 protein structure for the first time, verifying that the classic structural and conformational characteristics of the PP1 regulatory subunits are maintained. Our review is especially focused on finding evidence linking IIIG9 dysfunction with the course of some pathologies, such as ciliopathies, drug dependence, diseases based on neurological development, and the development of specific high-malignancy and -frequency brain tumors in the pediatric population. Finally, we propose that IIIG9 is a relevant regulator of PP1 function in physiological and pathological processes in the CNS.

Circulating ghrelin crosses the blood-cerebrospinal fluid barrier via growth hormone secretagogue receptor dependent and independent mechanisms.

Ghrelin is a peptide hormone mainly secreted from gastrointestinal tract that acts via the growth hormone secretagogue receptor (GHSR), which is highly expressed in the brain. Strikingly, the accessibility of ghrelin to the brain seems to be limited and restricted to few brain areas. Previous studies in mice have shown that ghrelin can access the brain via the blood-cerebrospinal fluid (CSF) barrier, an interface constituted by the choroid plexus and the hypothalamic tanycytes. Here, we performed a variety of in vivo and in vitro studies to test the hypothesis that the transport of ghrelin across the blood-CSF barrier occurs in a GHSR-dependent manner. In vivo, we found that the uptake of systemically administered fluorescent ghrelin in the choroid plexus epithelial (CPE) cells and in hypothalamic tanycytes depends on the presence of GHSR. Also, we detected lower levels of CSF ghrelin after a systemic ghrelin injection in GHSR-deficient mice, as compared to WT mice. In vitro, the internalization of fluorescent ghrelin was reduced in explants of choroid plexus from GHSR-deficient mice, and unaffected in primary cultures of hypothalamic tanycytes derived from GHSR-deficient mice. Finally, we found that the GHSR mRNA is detected in a pool of CPE cells, but is nearly undetectable in hypothalamic tanycytes with current approaches. Thus, our results suggest that circulating ghrelin crosses the blood-CSF barrier mainly by a mechanism that involves the GHSR, and also possibly via a GHSR-independent mechanism.

The ependymal cell cytoskeleton in the normal and injured spinal cord of mice.

The cytoskeleton of ependymal cells is fundamental to organize and maintain the normal architecture of the central canal (CC). However, little is known about the plasticity of cytoskeletal components after spinal cord injury. Here, we focus on the structural organization of the cytoskeleton of ependymal cells in the normal and injured spinal cord of mice (both females and males) using immunohistochemical and electron microscopy techniques. We found that in uninjured animals, the actin cytoskeleton (as revealed by phalloidin staining) was arranged following the typical pattern of polarized epithelial cells with conspicuous actin pools located in the apical domain of ependymal cells. Transmission electron microscopy images showed microvilli tufts, long cilia, and characteristic intercellular membrane specializations. After spinal cord injury, F-actin rearrangements paralleled by fine structural modifications of the apical domain of ependymal cells were observed. These changes involved disruptions of the apical actin pools as well as fine structural modifications of the microvilli tufts. When comparing the control and injured spinal cords, we also found modifications in the expression of vimentin and glial fibrillary acidic protein (GFAP). After injury, vimentin expression disappeared from the most apical domains of ependymal cells but the number of GFAP-expressing cells within the CC increased. As in other polarized epithelia, the plastic changes in the cytoskeleton may be critically involved in the reaction of ependymal cells following a traumatic injury of the spinal cord.

Cellular composition and organization of the spinal cord central canal during metamorphosis of the frog Xenopus laevis.

Studying the cellular composition and morphological changes of cells lining the central canal during Xenopus laevis metamorphosis could contribute to understand postnatal development and spinal cord regeneration. Here we report the analysis of central canal cells at different stages during metamorphosis using immunofluorescence for protein markers expression, transmission and scanning electron microscopy and cell proliferation assays. The central canal was regionalized according to expression of glial markers, ultrastructure, and proliferation in dorsal, lateral, and ventral domains with differences between larvae and froglets. In regenerative larvae, all cell types were uniciliated, have a radial morphology, and elongated nuclei with lax chromatin, resembling radial glial cells. Important differences in cells of nonregenerative froglets were observed, although uniciliated cells were found, the most abundant cells had multicilia and revealed extensive changes in the maturation and differentiation state. The majority of dividing cells in larvae corresponded to uniciliated cells at dorsal and lateral domains in a cervical-lumbar gradient, correlating with undifferentiated features. Neurons contacting the lumen of the central canal were detected in both stages and revealed extensive changes in the maturation and differentiation state. However, in froglets a very low proportion of cells incorporate 5-ethynyl-2'-deoxyuridine (EdU), associated with the differentiated profile and with the increase of multiciliated cells. Our work showed progressive changes in the cell types lining the central canal of Xenopus laevis spinal cord which are correlated with the regenerative capacities.

A simple strategy for culturing morphologically-conserved rat hypothalamic tanycytes.

Hypothalamic tanycytes are specialized bipolar ependymal cells that line the floor of the third ventricle. Given their strategic location, tanycytes are believed to play several key functions including being a selective barrier and controlling the amount of hypothalamic-derived factors reaching the anterior pituitary. The in vitro culture of these cells has proved to be difficult. Here, we report an improved method for the generation of primary cultures of rat hypothalamic tanycytes. Ependymal cultures were derived from tissue dissected out of the median eminence region of 10-day-old rats and cultured in a chemically defined medium containing DMEM:F12, serum albumin, insulin, transferrin and the antibiotic gentamycin. After 7 days in vitro, ∼30% of the cultured cells exhibited morphological features of tanycytes as observed by phase contrast or scanning electron microscopy. Tanycyte-like cells were strongly immuno-reactive for vimentin and dopamine-cAMP-regulated phospho-protein (DARPP-32) and weakly immune-reactive for glial fibrillary acidic protein. Tanycyte-like cells displayed a stable negative resting plasma membrane potential and failed to show spiking properties in response to current injections. When exposed to fluorescent beads in the culture medium, tanycyte-like cells exhibited a robust endocytosis. Thus, the present method effectively yields cultures containing tanycyte-like cells that resemble in vivo tanycytes in terms of morphologic features and molecular markers as well as electrical and endocytic activity. To our knowledge, this is the first protocol that allows the culturing of tanycyte-like cells that can be individually identified and that conserve the morphology of tanycytes in their natural physiological environment.

Analysis of brain nuclei accessible to ghrelin present in the cerebrospinal fluid.

Ghrelin is a stomach-derived peptide hormone that acts in the brain to regulate many important physiological functions. Ghrelin receptor, named the growth hormone secretagogue receptor (GHSR), is present in many brain areas with or without obvious direct access to ghrelin circulating in the bloodstream. Ghrelin is also present in the cerebrospinal fluid (CSF) but the brain targets of CSF ghrelin are unclear. Here, we studied which brain areas are accessible to ghrelin present in the CSF. For this purpose, we centrally injected mice with fluorescein-labeled ghrelin (F-ghrelin) peptide tracer and then systematically mapped the distribution of F-ghrelin signal through the brain. Our results indicated that centrally injected F-ghrelin labels neurons in most of the brain areas where GHSR is present. Also, we detected F-ghrelin uptake in the ependymal cells of both wild-type and GHSR-null mice. We conclude that CSF ghrelin is able to reach most of brain areas expressing GHSR. Also, we propose that the accessibility of CSF ghrelin to the brain parenchyma occurs through the ependymal cells in a GHSR-independent manner.

SVCT2 vitamin C transporter expression in progenitor cells of the postnatal neurogenic niche.

Known as a critical antioxidant, recent studies suggest that vitamin C plays an important role in stem cell generation, proliferation and differentiation. Vitamin C also enhances neural differentiation during cerebral development, a function that has not been studied in brain precursor cells. We observed that the rat neurogenic niche is structurally organized at day 15 of postnatal development, and proliferation and neural differentiation increase at day 21. In the human brain, a similar subventricular niche was observed at 1-month of postnatal development. Using immunohistochemistry, sodium-vitamin C cotransporter 2 (SVCT2) expression was detected in the subventricular zone (SVZ) and rostral migratory stream (RMS). Low co-distribution of SVCT2 and ßIII-tubulin in neuroblasts or type-A cells was detected, and minimal co-localization of SVCT2 and GFAP in type-B or precursor cells was observed. Similar results were obtained in the human neurogenic niche. However, BrdU-positive cells also expressed SVCT2, suggesting a role of vitamin C in neural progenitor proliferation. Primary neurospheres prepared from rat brain and the P19 teratocarcinoma cell line, which forms neurospheres in vitro, were used to analyze the effect of vitamin C in neural stem cells. Both cell types expressed functional SVCT2 in vitro, and ascorbic acid (AA) induced their neural differentiation, increased ßIII-tubulin and SVCT2 expression, and amplified vitamin C uptake.