Anatomical study of serotonergic innervation and 5-HT1A receptor in the human spinal cord

Serotonergic innervation of the spinal cord in mammals has multiple roles in the control of motor, sensory and visceral functions. In rats, functional consequences of spinal cord injury at thoracic level can be improved by a substitutive transplantation of serotonin (5-HT) neurons or regeneration under the trophic influence of grafted stem cells. Translation to either pharmacological and/or cellular therapies in humans requires the mapping of the spinal cord 5-HT innervation and its receptors to determine their involvement in specific functions. Here, we have performed a preliminary mapping of serotonergic processes and serotonin-lA (5-HT1A) receptors in thoracic and lumbar segments of the human spinal cord. As in rodents and non-human primates, 5-HT profiles in human spinal cord are present in the ventral horn, surrounding motoneurons, and also contact their presumptive dendrites at lumbar level. 5-HT1A receptors are present in the same area, but are more densely expressed at lumbar level. 5-HT profiles are also present in the intermediolateral region, where 5-HT1A receptors are absent. Finally, we observed numerous serotonergic profiles in the superficial part (equivalent of Rexed lamina II) of the dorsal horn, which also displayed high levels of 5-HT1A receptors. These findings pave the way for local specific therapies involving cellular and/or pharmacological tools targeting the serotonergic system

Glioma stem cells invasive phenotype at optimal stiffness is driven by MGAT5 dependent mechanosensing.

BACKGROUND: Glioblastomas stem-like cells (GSCs) by invading the brain parenchyma, remains after resection and radiotherapy and the tumoral microenvironment become stiffer. GSC invasion is reported as stiffness sensitive and associated with altered N-glycosylation pattern. Glycocalyx thickness modulates integrins mechanosensing, but details remain elusive and glycosylation enzymes involved are unknown. Here, we studied the association between matrix stiffness modulation, GSC migration and MGAT5 induced N-glycosylation in fibrillar 3D context. METHOD: To mimic the extracellular matrix fibrillar microenvironments, we designed 3D-ex-polyacrylonitrile nanofibers scaffolds (NFS) with adjustable stiffnesses by loading multiwall carbon nanotubes (MWCNT). GSCs neurosphere were plated on NFSs, allowing GSCs migration and MGAT5 was deleted using CRISPR-Cas9. RESULTS: We found that migration of GSCs was maximum at 166 kPa. Migration rate was correlated with cell shape, expression and maturation of focal adhesion (FA), Epithelial to Mesenchymal Transition (EMT) proteins and (β1,6) branched N-glycan binding, galectin-3. Mutation of MGAT5 in GSC inhibited N-glycans (β1-6) branching, suppressed the stiffness dependence of migration on 166 kPa NFS as well as the associated FA and EMT protein expression. CONCLUSION: MGAT5 catalysing multibranched N-glycans is a critical regulators of stiffness induced invasion and GSCs mechanotransduction, underpinning MGAT5 as a serious target to treat cancer

Isolation of mineralizing Nestin+ Nkx6.1+ vascular muscular cells from the adult human spinal cord

<p>Abstract</p> <p>Background</p> <p>The adult central nervous system (CNS) contains different populations of immature cells that could possibly be used to repair brain and spinal cord lesions. The diversity and the properties of these cells in the human adult CNS remain to be fully explored. We previously isolated Nestin⁺Sox2⁺neural multipotential cells from the adult human spinal cord using the neurosphere method (i.e. non adherent conditions and defined medium).</p> <p>Results</p> <p>Here we report the isolation and long term propagation of another population of Nestin⁺cells from this tissue using adherent culture conditions and serum. QPCR and immunofluorescence indicated that these cells had mesenchymal features as evidenced by the expression of Snai2 and Twist1 and lack of expression of neural markers such as Sox2, Olig2 or GFAP. Indeed, these cells expressed markers typical of smooth muscle vascular cells such as Calponin, Caldesmone and Acta2 (Smooth muscle actin). These cells could not differentiate into chondrocytes, adipocytes, neuronal and glial cells, however they readily mineralized when placed in osteogenic conditions. Further characterization allowed us to identify the Nkx6.1 transcription factor as a marker for these cells. Nkx6.1 was expressed in vivo by CNS vascular muscular cells located in the parenchyma and the meninges.</p> <p>Conclusion</p> <p>Smooth muscle cells expressing Nestin and Nkx6.1 is the main cell population derived from culturing human spinal cord cells in adherent conditions with serum. Mineralization of these cells in vitro could represent a valuable model for studying calcifications of CNS vessels which are observed in pathological situations or as part of the normal aging. In addition, long term propagation of these cells will allow the study of their interaction with other CNS cells and their implication in scar formation during spinal cord injury.</p

Cholinergic Enhancement of Cell Proliferation in the Postnatal Neurogenic Niche of the Mammalian Spinal Cord

The region surrounding the central canal (CC) of the spinal cord is a highly plastic area, defined as a postnatal neurogenic niche. Within this region are ependymal cells which can proliferate and differentiate to form new astrocytes and oligodendrocytes following injury and cerebrospinal fluid contacting cells (CSFcCs). The specific environmental conditions, including the modulation by neurotransmitters that influence these cells and their ability to proliferate, are unknown. Here we show that acetylcholine promotes the proliferation of ependymal cells in mice under both in vitro and in vivo conditions. Using whole cell patch clamp in acute spinal cord slices, acetylcholine directly depolarised ependymal cells and CSFcCs. Antagonism by specific nicotinic acetylcholine receptor (nAChR) antagonists or potentiation by the α7*nAChR modulator PNU 120596 revealed that both α7*nAChRs and non-α7*nAChRs mediated the cholinergic responses. Using the nucleoside analogue EdU (5-ethynyl-2'-deoxyuridine) as a marker of cell proliferation, application of α7*nAChR modulators in spinal cord cultures or in vivo induced proliferation in the CC region, producing Sox-2 expressing ependymal cells. Proliferation also increased in the white and grey matter. PNU 120596 administration also increased the proportion of cells co-expressing oligodendrocyte markers. Thus, variation in the availability of acetylcholine can modulate the rate of proliferation of cells in the ependymal cell layer and white 1 and grey matter through α7*nAChRs. This study highlights the need for further investigation into how neurotransmitters regulate the response of the spinal cord to injury or during aging

The adult spinal cord harbors a population of GFAP-positive progenitors with limited self-renewal potential

Adult neural stem cells (aNSCs) of the forebrain are GFAP-expressing cells that are intercalated within ependymal cells of the subventricular zone (SVZ). Cells showing NSCs characteristics in vitro can also be isolated from the periaqueductal region in the adult spinal cord (SC), but contradicting results exist concerning their glial versus ependymal identity. We used an induci- ble transgenic mouse line (hGFAP-CreERT2) to conditionally label GFAP-expressing cells in the adult SVZ and SC periaque- duct, and directly and systematically compared their self-renewal and multipotential properties in vitro. We demonstrate that a population of GFAP1 cells that share the morphology and the antigenic properties of SVZ-NSCs mostly reside in the dorsal aspect of the central canal (CC) throughout the spinal cord. These cells are non-proliferative in the intact spinal cord, but incorporate the S-phase marker EdU following spinal cord injury. Multipotent, clonal YFP-expressing neurospheres (i.e., deriv- ing from recombined GFAP-expressing cells) were successfully obtained from both the intact and injured spinal cord. These spheres however showed limited self-renewal properties when compared with SVZ-neurospheres, even after spinal cord injury. Altogether, these results demonstrate that significant differences exist in NSCs lineages between neurogenic and non- neurogenic regions of the adult CNS. Thus, although we confirm that a population of multipotent GFAP1 cells co-exists alongside with multipotent ependymal cells within the adult SC, we identify these cells as multipotent progenitors showing limited self-renewal properties

ATF3 is a novel nuclear marker for migrating ependymal stem cells in the rat spinal cord

The present study identified ATF3 as a novel dynamic marker for ependymal stem/progenitor cells (nestin, vimentin and SOX2 positive) around the central canal of the neonatal or adult rat spinal cord. While quiescent ependymal cells showed cytoplasmic ATF3 expression, during 6-24. h in vitro these cells mobilized and acquired intense nuclear ATF3 staining. Their migratory pattern followed a centrifugal pathway toward the dorsal and ventral funiculi, reminiscent of the rostral migratory stream of the brain subventricular stem cells. Thus, the chain cell formation was, by analogy, termed funicular migratory stream (FMS). The FMS process preceded the strong proliferation of ependymal cells occurring only after 24. h in vitro. Pharmacological inhibition of MAPK-p38 and JNK/c-Jun (upstream effectors of ATF3 activation) prevented the FMS mobilization of ATF3 nuclear-positive cells. Excitotoxicity or ischemia-like conditions, reported to evoke neuronal and glial injury, did not further enhance migration of ependymal cells at 24. h, suggesting that, at this early stage of damage, the FMS phenomenon had peaked and that more extensive repair processes are delayed beyond this time point. ATF3 is, therefore, useful to identify activation and migration of endogenous stem cells of the rat spinal cord in vitro. \ua9 2014

Excitotoxic cell death induces delayed proliferation of endogenous neuroprogenitor cells in organotypic slice cultures of the rat spinal cord

The aim of the present report was to investigate whether, in the mammalian spinal cord, cell death induced by transient excitotoxic stress could trigger activation and proliferation of endogenous neuroprogenitor cells as a potential source of a lesion repair process and the underlying time course. Because it is difficult to address these issues in vivo, we used a validated model of spinal injury based on rat organotypic slice cultures that retain the fundamental tissue cytoarchitecture and replicate the main characteristics of experimental damage to the whole spinal cord. Excitotoxicity evoked by 1 h kainate application produced delayed neuronal death (40%) peaking after 1 day without further losses or destruction of white matter cells for up to 2 weeks. After 10 days, cultures released a significantly larger concentration of endogenous glutamate, suggesting functional network plasticity. Indeed, after 1 week the total number of cells had returned to untreated control level, indicating substantial cell proliferation. Activation of progenitor cells started early as they spread outside the central area, and persisted for 2 weeks. Although expression of the neuronal progenitor phenotype was observed at day 3, peaked at 1 week and tapered off at 2 weeks, very few cells matured to neurons. Astroglia precursors started proliferating later and matured at 2 weeks. These data show insult-related proliferation of endogenous spinal neuroprogenitors over a relatively brief time course, and delineate a narrow temporal window for future experimental attempts to drive neuronal maturation and for identifying the factors regulating this process. \ua9 2013 Macmillan Publishers Limited. All rights reserved