Clarissa Schitine

Astrocyte heterogeneity in the brain: from development to disease

In the last decades, astrocytes have risen from passive supporters of neuronal activity to central players in brain function and cognition. Likewise, the heterogeneity of astrocytes starts to become recognized in contrast to the homogeneous population previously predicted. In this review, we focused on astrocyte heterogeneity in terms of their morphological, protein expression and functional aspects, and debate in a historical perspective the diversity encountered in glial progenitors and how they may reflect mature astrocyte heterogeneity. We discussed data that show that different progenitors may have unsuspected roles in developmental processes. We have approached the functions of astrocyte subpopulations on the onset of psychiatric and neurological diseases.

Activation of type 1 cannabinoid receptor (CB1R) promotes neurogenesis in murine subventricular zone cell cultures.

The endocannabinoid system has been implicated in the modulation of adult neurogenesis. Here, we describe the effect of type 1 cannabinoid receptor (CB1R) activation on self-renewal, proliferation and neuronal differentiation in mouse neonatal subventricular zone (SVZ) stem/progenitor cell cultures. Expression of CB1R was detected in SVZ-derived immature cells (Nestin-positive), neurons and astrocytes. Stimulation of the CB1R by (R)-(+)-Methanandamide (R-m-AEA) increased self-renewal of SVZ cells, as assessed by counting the number of secondary neurospheres and the number of Sox2+/+ cell pairs, an effect blocked by Notch pathway inhibition. Moreover, R-m-AEA treatment for 48 h, increased proliferation as assessed by BrdU incorporation assay, an effect mediated by activation of MAPK-ERK and AKT pathways. Surprisingly, stimulation of CB1R by R-m-AEA also promoted neuronal differentiation (without affecting glial differentiation), at 7 days, as shown by counting the number of NeuN-positive neurons in the cultures. Moreover, by monitoring intracellular calcium concentrations ([Ca2+]i) in single cells following KCl and histamine stimuli, a method that allows the functional evaluation of neuronal differentiation, we observed an increase in neuronal-like cells. This proneurogenic effect was blocked when SVZ cells were co-incubated with R-m-AEA and the CB1R antagonist AM 251, for 7 days, thus indicating that this effect involves CB1R activation. In accordance with an effect on neuronal differentiation and maturation, R-m-AEA also increased neurite growth, as evaluated by quantifying and measuring the number of MAP2-positive processes. Taken together, these results demonstrate that CB1R activation induces proliferation, self-renewal and neuronal differentiation from mouse neonatal SVZ cell cultures.

Galanin promotes neuronal differentiation in murine subventricular zone cell cultures.

Neural stem cells of the subventricular zone (SVZ) represent a potentially important source of surrogate cells for the treatment of brain damage. Proper use of these cells for neuronal replacement depends on the ability to drive neuronal differentiation. Several neuromodulators stimulate neurogenesis. Here we examined the effects of the neuropeptide galanin, on neuronal differentiation in murine SVZ cultures. SVZ neurospheres obtained from early postnatal mice were treated with 10 nM to 2 μM galanin. Galanin promoted neuronal differentiation, increasing numbers of NeuN-, vesicular GABA transporter- and tyrosine hydroxylase-expressing neurons. In contrast, galanin neither affected cell proliferation assessed by BrdU incorporation nor cell death evaluated by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). Neuronal differentiation was further confirmed at the functional level by measuring [Ca(2+)]i variations in single SVZ cells after KCl and histamine stimulations to distinguish neurons from immature cells. Galanin treatment increased the numbers of neuronal-like responding cells compared to immature cells. Using selective agonists (M617, AR-M1896) and antagonists (galantide, M871) for galanin receptors 1 and 2, we showed that both galanin receptors mediated neuronal differentiation. Early proneuronal effects of galanin included positive regulation of the transcription factor neurogenin-1 (Ngn1). In addition, galanin promoted axonogenesis and dendritogenesis, increasing both the length of phosphorylated stress-activated protein kinase- and Tau-positive axons and the numbers of microtubule associated protein-2 (MAP-2)-labelled dendrites. Moreover, galanin inhibited SVZ cell migration in the transwell assay. Our results show a proneurogenic effect of galanin and open new perspectives for future applications in stem cell-based therapies for neuronal replacement.

Ampakine CX546 increases proliferation and neuronal differentiation in subventricular zone stem/progenitor cell cultures

Ampakines are chemical compounds known to modulate the properties of ionotropic α‐amino‐3‐hydroxyl‐5‐methyl‐4‐isoxazole‐propionate (AMPA)‐subtype glutamate receptors. The functional effects attributed to ampakines involve plasticity and the increase in synaptic efficiency of neuronal circuits, a process that may be intimately associated with differentiation of newborn neurons. The subventricular zone (SVZ) is the main neurogenic niche of the brain, containing neural stem cells with brain repair potential. Accordingly, the identification of new pharmaceutical compounds with neurogenesis‐enhancing properties is important as a tool to promote neuronal replacement based on the use of SVZ cells. The purpose of the present paper is to examine the possible proneurogenic effects of ampakine CX546 in cell cultures derived from the SVZ of early postnatal mice. We observed that CX546 (50 μm) treatment triggered an increase in proliferation, evaluated by BrdU incorporation assay, in the neuroblast lineage. Moreover, by using a cell viability assay (TUNEL) we found that, in contrast to AMPA, CX546 did not cause cell death. Also, both AMPA and CX546 stimulated neuronal differentiation as evaluated morphologically through neuronal nuclear protein (NeuN) immunocytochemistry and functionally by single‐cell calcium imaging. Accordingly, short exposure to CX546 increased axonogenesis, as determined by the number and length of tau‐positive axons co‐labelled for the phosphorylated form of SAPK/JNK (P‐JNK), and dendritogenesis (MAP2‐positive neurites). Altogether, this study shows that ampakine CX546 promotes neurogenesis in SVZ cell cultures and thereby may have potential for future stem cell‐based therapies.

Functional Identification of Cell Phenotypes Differentiating from Mice Retinal Neurospheres Using Single Cell Calcium Imaging

Degeneration of neural retina causes vision impairment and can lead to blindness. Neural stem and progenitor cells might be used as a tool directed to regenerative medicine of the retina. Here, we describe a novel platform for cell phenotype-specific drug discovery and screening of proneurogenic factors, able to boost differentiation of neural retinal progenitor cells. By using single cell calcium imaging (SCCI) and a rational-based stimulation protocol, a diversity of cells emerging from differentiated retinal neurosphere cultures were identified. Exposure of retinal progenitor cultures to KCl or to α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) stimulated Ca2+ transients in microtubule-associated protein 2 (MAP-2) positive neurons. Doublecortin (DCX) and polysialated neural cell adhesion molecule (PSA-NCAM) positive neuroblasts were distinguished from differentiated neurons on the basis of their response to muscimol. Ca2+ fluxes in glial fibrillary acidic protein (GFAP) or glutamine synthetase (GS) positive cells were induced by ATP. To validate the platform, neurospheres were treated with brain-derived neurotrophic factor (BDNF) (proneurogenic) or ciliary neurotrophic factor (CNTF) (gliogenic factor). BDNF increased the percentage of differentiated cells expressing Tuj-1 sensitive to KCl or AMPA and reduced the population of cells responding to muscimol. CNTF exposure resulted in a higher number of cells expressing GFAP responding to ATP. All together, our data may open new perspectives for cell type-specific discovery of drug targets and screening of novel proneurogenic factors to boost differentiation of neural retina cells to treat degenerative retinal diseases.

GABA uptake by purified avian Müller glia cells in culture.

GABA is the main inhibitory aminoacid transmitter present in neurons and glial cells. Its uptake is carried out by specific high-affinity Na+/Cl− dependent transporters (GATs). It has been reported in the past that, in the avian retina, [3H]GABA appears to be exclusively accumulated by horizontal and amacrine cells in the inner nuclear layer, and also by ganglion cells. Purified chick Müller glia cultures were able to take up [3H]GABA in a Na+ and Cl− dependent way. Increasing GABA concentration increases GABA uptake by these cells, reaching half-maximal transport efficiency (EC50) around 0.3 mM. [3H]GABA uptake by Müller glia neuronal-free cultures was not mediated by neuronal transporters since it was not blocked by NNC-711, but was inhibited by beta-alanine, a specific glial transporter inhibitor. Chick Müller glia in culture express both GAT-1 and GAT-3 GABA transporters. Although mixed neuron-glial dense cultures released GABA upon glutamate, high K+ or veratridine stimulation, Müller glial cells did not release [3H]GABA upon treatment with these agents, suggesting that different from neurons, transporter mediated GABA release is not a common mechanism operating in these cells. The data also suggest that Müller cells take up GABA unidirectionally, which may constitute an important mechanism of inactivating GABA activity mediated by neurons.

Modulation of subventricular zone oligodendrogenesis: a role for hemopressin?

Neural stem cells (NSCs) from the subventricular zone (SVZ) have been indicated as a source of new oligodendrocytes to use in regenerative medicine for myelin pathologies. Indeed, NSCs are multipotent cells that can self-renew and differentiate into all neural cell types of the central nervous system. In normal conditions, SVZ cells are poorly oligodendrogenic, nevertheless their oligodendrogenic potential is boosted following demyelination. Importantly, progressive restriction into the oligodendrocyte fate is specified by extrinsic and intrinsic factors, endocannabinoids being one of these factors. Although a role for endocannabinoids in oligodendrogenesis has already been foreseen, selective agonists and antagonists of cannabinoids receptors produce severe adverse side effects. Herein, we show that hemopressin (Hp), a modulator of CB1 receptors, increased oligodendroglial differentiation in SVZ neural stem/progenitor cell cultures derived from neonatal mice. The original results presented in this work suggest that Hp and derivates may be of potential interest for the development of future strategies to treat demyelinating diseases.

Functional plasticity of GAT-3 in avian Müller cells is regulated by neurons via a glutamatergic input

GABA (γ-amino butyric acid) is the major inhibitory transmitter in the central nervous system and its action is terminated by specific transporters (GAT), found in neurons and glial cells. We have previously described that GAT-3 is responsible for GABA uptake activity in cultured avian Müller cells and that it operates in a Na(+) and Cl(-) dependent manner. Here we show that glutamate decreases [(3)H] GABA uptake in purified cultured glial cells up to 50%, without causing cell death. This effect is mediated by ionotropic glutamatergic receptors. Glutamate inhibition on GABA uptake is not reverted by inhibitors of protein kinase C or modified by agents that modulate cyclic AMP/PKA. Biotinylation experiments demonstrate that this reduction in GABA uptake correlates with a decrease in GAT-3 plasma membrane levels. Interestingly, both GAT-1 and GAT-3 mRNA levels are also decreased by glutamate. Conditioned media (CM) prepared from retinal neurons could also decrease GABA influx, and glutamate receptor antagonists (MK-801 + CNQX) were able to prevent this effect. However, glutamate levels in CM were not different from those found in fresh media, indicating that a glutamatergic co-agonist or modulator could be regulating GABA uptake by Müller cells in this scenario. In the whole avian retina, GAT-3 is present from embryonic day 5 (E5) increasing up to the end of embryonic development and post-hatch period exclusively in neuronal layers. However, this pattern may change in pathological conditions, which drive GAT-3 expression in Müller cells. Our data suggest that in purified cultures and upon extensive neuronal lesion in vivo, shown as a Brn3a reduced neuronal cells and an GFAP increased gliosis, Müller glia may change its capacity to take up GABA due to GAT-3 up regulation and suggests a regulatory interplay mediated by glutamate between neurons and glial cells in this process.

Neuro-glial cannabinoid receptors modulate signaling in the embryonic avian retina

&NA; Endocannabinoids are endogenous lipids that activate selective G protein coupled receptors (CB1 and CB2), mostly found at neuronal presynaptic sites in the nervous system. One of the main consequences of the activation of CB receptors is a decrease in GABA or glutamate release, controlling cell excitability. Here we studied the expression of CB1 and CB2 receptors in E8C8 cultured retina cells (embryonic day 8 and 8 days in vitro) using immunocytochemistry and western blot analysis. We also evaluated their functions in terms of cyclic AMP (cAMP) production, single cell calcium imaging (SCCI) and GABA release induced in basal conditions or activated by l‐Aspartate (L‐ASP) in cell cultures or under ischemia in young chick retina. We show that both cannabinoid receptors are expressed in retinal neurons and glial cells. WIN 55,212‐2 (WIN, a CB1/CB2 agonist) decreased cAMP production in cultured avian embryonic retinal cells in basal conditions. WIN also led to a decrease in the number of glial cells that increased Ca2+ levels evoked by ATP, but had no effect in Ca2+ shifts in neuronal cells activated by KCl. Finally, WIN inhibited [3H]‐GABA release induced by KCl or L‐ASP, accumulated in amacrine cells, but had no effect in the amount of GABA released in an oxygen glucose deprivation (OGD) condition. Altogether, our data indicate that cannabinoid receptors function as regulators of avian retina signaling at critical embryonic stages during synapse formation. HighlightsBoth CB1 and CB2 are expressed in retinal neurons and glial cells in culture.WIN 55,212‐2 decreases cAMP production in retinal cells in basal conditions.WIN decreases the number of glial cells that increased Ca2+ evoked by ATP.WIN inhibited [3H]‐GABA release induced by KCl or L‐Aspartate.WIN had no effect in GABA released in an oxygen glucose deprivation condition in chick retina.

Beta-adrenergic receptor activation increases GABA uptake in adolescent mice frontal cortex: Modulation by cannabinoid receptor agonist WIN55,212-2

&NA; GABA transporters regulate synaptic GABA levels and dysfunctions in this system might result in psychiatric disorders. The endocannabinoid system (ECS) is the main circuit breaker in the nervous system and may alter noradrenaline (NA) communication, which in turn modulates the release of GABA. However, a close relationship between these systems has not been recognized. We asked whether NA and ECS might control extracellular GABA levels in slices of frontal cortex (FC) of adolescent Swiss mice with 40 days after birth (PN40). Here we show that NA and isoproterenol (ISO), a beta‐adrenergic agonist, increased [3H]‐GABA uptake in mice FC, while alpha1‐adrenergic agonist phenylephrine had no effect. As GAT‐1 is expressed and fully functional at the FC, addition of NO‐711, a GAT‐1 inhibitor, dose dependently blocked [3H]‐GABA uptake. The increase of [3H]‐GABA uptake induced by ISO was also blocked by NO‐711. [3H]‐GABA release induced by 80 mM KCl was reduced by NO‐711, but not by removal of Ca2+. ISO also increased cyclic AMP (cAMP) levels and addition of WIN 55,212‐2, a mixed CB1/CB2 receptor agonist, inhibited the effect of ISO in GABA uptake increase, GAT‐1 expression and cAMP levels compared to control. Our data show that GABA transport increased by NA and ISO is negatively regulated by cannabinoid receptor agonist WIN55,212‐2. HighlightsNoradrenaline increases [3H]‐GABA uptake in adolescent mice Frontal Cortex.The increase of [3H]‐GABA uptake induced by isoproterenol (ISO) is blocked by NO‐711.[3H]‐GABA release induced by 80 mM KCl is reduced by NO‐711, but not by removal of Ca2+.WIN 55,212‐2 inhibited the effect of ISO in GABA uptake, GAT‐1 expression and cAMP levels.