Recreating mouse cortico-hippocampal neuronal circuit in microfluidic devices to study BDNF axonal transport upon glucocorticoid treatment.

BDNF levels are reduced in the chronically stressed brain, in the area of hippocampus. Part of the hippocampal BDNF is provided by neuronal projection of the entorhinal cortex. Studying the cortico-hippocampal transport of BDNF in vivo is technically difficult. Here, we describe a protocol that reproduces mouse cortico-hippocampal circuit in vitro by plating neurons on the microfluidic devices and infecting the neurons with virus-encoding BDNF-mCherry, which allows investigation of the effects of elevated corticosterone levels on BDNF axonal transport. For complete details on the use and execution of this protocol, please refer to Agasse et al. (2020).

Chronic Corticosterone Elevation Suppresses Adult Hippocampal Neurogenesis by Hyperphosphorylating Huntingtin.

Chronic exposure to stress is a major risk factor for neuropsychiatric disease, and elevated plasma corticosterone (CORT) correlates with reduced levels of both brain-derived neurotrophic factor (BDNF) and hippocampal neurogenesis. Precisely how these phenomena are linked, however, remains unclear. Using a cortico-hippocampal network-on-a-chip, we find that the glucocorticoid receptor agonist dexamethasone (DXM) stimulates the cyclin-dependent kinase 5 (CDK5) to phosphorylate huntingtin (HTT) at serines 1181 and 1201 (S1181/1201), which retards BDNF vesicular transport in cortical axons. Parallel studies in mice show that CORT induces phosphorylation of these same residues, reduces BDNF levels, and suppresses neurogenesis. The adverse effects of CORT are reduced in mice bearing an unphosphorylatable mutant HTT (HdhS1181A/S1201A). The protective effect of unphosphorylatable HTT, however, disappears if neurogenesis is blocked. The CDK5-HTT pathway, which regulates BDNF transport in the cortico-hippocampal network, thus provides a missing link between elevated CORT levels and suppressed neurogenesis.

Huntington's disease alters human neurodevelopment.

Although Huntington's disease is a late-manifesting neurodegenerative disorder, both mouse studies and neuroimaging studies of presymptomatic mutation carriers suggest that Huntington's disease might affect neurodevelopment. To determine whether this is actually the case, we examined tissue from human fetuses (13 weeks gestation) that carried the Huntington's disease mutation. These tissues showed clear abnormalities in the developing cortex, including mislocalization of mutant huntingtin and junctional complex proteins, defects in neuroprogenitor cell polarity and differentiation, abnormal ciliogenesis, and changes in mitosis and cell cycle progression. We observed the same phenomena in Huntington's disease mouse embryos, where we linked these abnormalities to defects in interkinetic nuclear migration of progenitor cells. Huntington's disease thus has a neurodevelopmental component and is not solely a degenerative disease.

Heterocellular Contacts with Mouse Brain Endothelial Cells Via Laminin and α6ß1 Integrin Sustain Subventricular Zone (SVZ) Stem/Progenitor Cells Properties.

Neurogenesis in the subventricular zone (SVZ) is regulated by diffusible factors and cell-cell contacts. In vivo, SVZ stem cells are associated with the abluminal surface of blood vessels and such interactions are thought to regulate their neurogenic capacity. SVZ neural stem cells (NSCs) have been described to contact endothelial-derived laminin via α6ß1 integrin. To elucidate whether heterocellular contacts with brain endothelial cells (BEC) regulate SVZ cells neurogenic capacities, cocultures of SVZ neurospheres and primary BEC, both obtained from C57BL/6 mice, were performed. The involvement of laminin-integrin interactions in SVZ homeostasis was tested in three ways. Firstly, SVZ cells were analyzed following incubation of BEC with the protein synthesis inhibitor cycloheximide (CHX) prior to coculture, a treatment expected to decrease membrane proteins. Secondly, SVZ cells were cocultured with BEC in the presence of an anti-α6 integrin neutralizing antibody. Thirdly, BEC were cultured with ß1-/- SVZ cells. We showed that contact with BEC supports, at least in part, proliferation and stemness of SVZ cells, as evaluated by the number of BrdU positive (+) and Sox2+ cells in contact with BEC. These effects are dependent on BEC-derived laminin binding to α6ß1 integrin and are decreased in cocultures incubated with anti-α6 integrin neutralizing antibody and in cocultures with SVZ ß1-/- cells. Moreover, BEC-derived laminin sustains stemness in SVZ cell cultures via activation of the Notch and mTOR signaling pathways. Our results show that BEC/SVZ interactions involving α6ß1 integrin binding to laminin, contribute to SVZ cell proliferation and stemness.

A novel drug conjugate, NEO212, targeting proneural and mesenchymal subtypes of patient-derived glioma cancer stem cells.

Glioblastoma multiforme (GBM), a highly malignant brain tumor, accounts for half of all gliomas. Despite surgery, radiation and chemotherapy, the median survival is between 12 and 15 months. The poor prognosis is due to tumor recurrence attributed to chemoresistant glioma cancer stem cells (GSCs). Here we examined the effects of a novel compound NEO212, which is composed of two covalently conjugated anti-cancer compounds - temozolomide (TMZ) and perillyl alcohol (POH), on GSCs expressing either the proneural or mesenchymal gene signatures. These GSCs were obtained from patient-derived tumor tissue. Our findings demonstrate that NEO212 is 10 fold more cytotoxic to GSCs than TMZ (standard-of-care). Furthermore, NEO212 is effective against both proneural and clinically aggressive mesenchymal GSC subtypes. The mechanism of NEO212 mediated-cytotoxicity is through double-strand DNA breaks and apoptosis. In vivo studies show that NEO212 significantly delays tumor growth of both proneural and mesenchymal tumor stem cell populations. Patient-derived GSCs and tumors derived from these cells are highly reflective of the heterogeneity in human GBM. The efficacy of NEO212 against both GSC subtypes indicates that NEO212 has great clinical potential to effectively target GBM.

Methamphetamine decreases dentate gyrus stem cell self-renewal and shifts the differentiation towards neuronal fate.

Methamphetamine (METH) is a highly addictive psychostimulant drug of abuse that negatively interferes with neurogenesis. In fact, we have previously shown that METH triggers stem/progenitor cell death and decreases neuronal differentiation in the dentate gyrus (DG). Still, little is known regarding its effect on DG stem cell properties. Herein, we investigate the impact of METH on mice DG stem/progenitor cell self-renewal functions. METH (10nM) decreased DG stem cell self-renewal, while 1nM delayed cell cycle in the G0/G1-to-S phase transition and increased the number of quiescent cells (G0 phase), which correlated with a decrease in cyclin E, pEGFR and pERK1/2 protein levels. Importantly, both drug concentrations (1 or 10nM) did not induce cell death. In accordance with the impairment of self-renewal capacity, METH (10nM) decreased Sox2(+)/Sox2(+) while increased Sox2(-)/Sox2(-) pairs of daughter cells. This effect relied on N-methyl-d-aspartate (NMDA) signaling, which was prevented by the NMDA receptor antagonist, MK-801 (10µM). Moreover, METH (10nM) increased doublecortin (DCX) protein levels consistent with neuronal differentiation. In conclusion, METH alters DG stem cell properties by delaying cell cycle and decreasing self-renewal capacities, mechanisms that may contribute to DG neurogenesis impairment followed by cognitive deficits verified in METH consumers.

Growth hormone pathways signaling for cell proliferation and survival in hippocampal neural precursors from postnatal mice.

BACKGROUND: Accumulating evidence suggests that growth hormone (GH) may play a major role in the regulation of postnatal neurogenesis, thus supporting the possibility that it may be also involved in promoting brain repair after brain injury. In order to gain further insight on this possibility, in this study we have investigated the pathways signaling the effect of GH treatment on the proliferation and survival of hippocampal subgranular zone (SGZ)-derived neurospheres. RESULTS: Our results demonstrate that GH treatment promotes both proliferation and survival of SGZ neurospheres. By using specific chemical inhibitors we have been also able to demonstrate that GH treatment promotes the activation of both Akt-mTOR and JNK signaling pathways, while blockade of these pathways either reduces or abolishes the GH effects. In contrast, no effect of GH on the activation of the Ras-ERK pathway was observed after GH treatment, despite blockade of this signaling path also resulted in a significant reduction of GH effects. Interestingly, SGZ cells were also capable of producing GH, and blockade of endogenous GH also resulted in a decrease in the proliferation and survival of SGZ neurospheres. CONCLUSIONS: Altogether, our findings suggest that GH treatment may promote the proliferation and survival of neural progenitors. This effect may be elicited by cooperating with locally-produced GH in order to increase the response of neural progenitors to adequate stimuli. On this view, the possibility of using GH treatment to promote neurogenesis and cell survival in some acquired neural injuries may be envisaged.

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.

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 ([Ca(2+)]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.

Polymeric nanoparticles to control the differentiation of neural stem cells in the subventricular zone of the brain.

Herein, we report the use of retinoic acid-loaded polymeric nanoparticles as a potent tool to induce the neuronal differentiation of subventricular zone neural stem cells. The intracellular delivery of retinoic acid by the nanoparticles activated nuclear retinoic acid receptors, decreased stemness, and increased proneurogenic gene expression. Importantly, this work reports for the first time a nanoparticle formulation able to modulate in vivo the subventricular zone neurogenic niche. The work further compares the dynamics of initial stages of differentiation between SVZ cells treated with retinoic acid-loaded polymeric nanoparticles and solubilized retinoic acid. The nanoparticle formulation developed here may ultimately offer new perspectives to treat neurodegenerative diseases.

Blockade of adenosine A2A receptors prevents interleukin-1ß-induced exacerbation of neuronal toxicity through a p38 mitogen-activated protein kinase pathway.

BACKGROUND AND PURPOSE: Blockade of adenosine A(2A) receptors (A(2A)R) affords robust neuroprotection in a number of brain conditions, although the mechanisms are still unknown. A likely candidate mechanism for this neuroprotection is the control of neuroinflammation, which contributes to the amplification of neurodegeneration, mainly through the abnormal release of pro-inflammatory cytokines such as interleukin(IL)-1ß. We investigated whether A(2A)R controls the signaling of IL-1ß and its deleterious effects in cultured hippocampal neurons. METHODS: Hippocampal neuronal cultures were treated with IL-1ß and/or glutamate in the presence or absence of the selective A(2A)R antagonist, SCH58261 (50 nmol/l). The effect of SCH58261 on the IL-1ß-induced phosphorylation of the mitogen-activated protein kinases (MAPKs) c-Jun N-terminal kinase (JNK) and p38 was evaluated by western blotting and immunocytochemistry. The effect of SCH58261 on glutamate-induced neurodegeneration in the presence or absence of IL-1ß was evaluated by nucleic acid and by propidium iodide staining, and by lactate dehydrogenase assay. Finally, the effect of A(2A)R blockade on glutamate-induced intracellular calcium, in the presence or absence of IL-1ß, was studied using single-cell calcium imaging. RESULTS: IL-1ß (10 to 100 ng/ml) enhanced both JNK and p38 phosphorylation, and these effects were prevented by the IL-1 type 1 receptor antagonist IL-1Ra (5 µg/ml), in accordance with the neuronal localization of IL-1 type 1 receptors, including pre-synaptically and post-synaptically. At 100 ng/ml, IL-1ß failed to affect neuronal viability but exacerbated the neurotoxicity induced by treatment with 100 µmol/l glutamate for 25 minutes (evaluated after 24 hours). It is likely that this resulted from the ability of IL-1ß to enhance glutamate-induced calcium entry and late calcium deregulation, both of which were unaffected by IL-1ß alone. The selective A(2A)R antagonist, SCH58261 (50 nmol/l), prevented both the IL-1ß-induced phosphorylation of JNK and p38, as well as the IL-1ß-induced deregulation of calcium and the consequent enhanced neurotoxicity, whereas it had no effect on glutamate actions. CONCLUSIONS: These results prompt the hypothesis that the neuroprotection afforded by A(2A)R blockade might result from this particular ability of A(2A)R to control IL-1ß-induced exacerbation of excitotoxic neuronal damage, through the control of MAPK activation and late calcium deregulation.

Histamine stimulates neurogenesis in the rodent subventricular zone.

Neural stem/progenitor cells present in the subventricular zone (SVZ) are a potential source of repairing cells after injury. Therefore, the identification of novel players that modulate neural stem cells differentiation can have a huge impact in stem cell-based therapies. Herein, we describe a unique role of histamine in inducing functional neuronal differentiation from cultured mouse SVZ stem/progenitor cells. This proneurogenic effect depends on histamine 1 receptor activation and involves epigenetic modifications and increased expression of Mash1, Dlx2, and Ngn1 genes. Biocompatible poly (lactic-co-glycolic acid) microparticles, engineered to release histamine in a controlled and prolonged manner, also triggered robust neuronal differentiation in vitro. Preconditioning with histamine-loaded microparticles facilitated neuronal differentiation of SVZ-GFP cells grafted in hippocampal slices and in in vivo rodent brain. We propose that neuronal commitment triggered by histamine per se or released from biomaterial-derived vehicles may represent a new tool for brain repair strategies.

Nanomedicine boosts neurogenesis: new strategies for brain repair.

The subventricular zone (SVZ) and the hippocampal subgranular zone (SGZ) comprise two main germinal niches in the adult mammalian brain. Within these regions there are self-renewing and multipotent neural stem cells (NSCs) which can ultimately give rise to new neurons, astrocytes and oligodendrocytes. Understanding how to efficiently trigger NSCs differentiation is crucial to devise new cellular therapies aimed to repair the damaged brain. A large amount of data ranging from epigenetic alterations, chromatin remodelling and signalling pathways involved in NSCs differentiation are now within reach. Furthermore, a vast array of proteins and molecules have been described to modulate NSCs fate and tested in innovative therapeutic applications, however with little success so far. Nowadays, the main focus is on how to manipulate these factors to our full advantage. Unfortunately, concerns related to solubility, stability, concentration or spatial and temporal positioning can hinder their desirable effects. Biomaterials emerge as the ideal support to overcome these limitations and consequently boost NSCs differentiation towards desired phenotypes. However, the balance between biomaterials and differentiating factors must be well established, since the bioaccumulation and concomitant toxicity can be an undesired side-effect. Currently, innovative materials and formulations including more degradable carriers allow a controlled and efficient release of bioactive factors with minimal side-effects. Recently, micro- and nanoparticles have been successfully used to deliver molecules able to induce neurogenesis. This review presents recent research that highlights the role of both extracellular environmental factors as well as molecular remodelling mechanisms in the control of NSCs differentiation processes. Appropriate biomaterials that may trigger an efficient delivery of therapeutic molecules will be also discussed. Therefore, the interface between NSCs biology and tissue engineering may offer great potential in future therapeutics for treatment or amelioration of neurodegenerative diseases or brain injury.

Histamine modulates microglia function.

BACKGROUND: Histamine is commonly acknowledged as an inflammatory mediator in peripheral tissues, leaving its role in brain immune responses scarcely studied. Therefore, our aim was to uncover the cellular and molecular mechanisms elicited by this molecule and its receptors in microglia-induced inflammation by evaluating cell migration and inflammatory mediator release. METHODS: Firstly, we detected the expression of all known histamine receptor subtypes (H1R, H2R, H3R and H4R), using a murine microglial cell line and primary microglia cell cultures from rat cortex, by real-time PCR analysis, immunocytochemistry and Western blotting. Then, we evaluated the role of histamine in microglial cell motility by performing scratch wound assays. Results were further confirmed using murine cortex explants. Finally, interleukin-1beta (IL-1ß) and tumor necrosis factor-alpha (TNF-α) levels were evaluated by ELISA measurements to determine the role of histamine on the release of these inflammatory mediators. RESULTS: After 12 h of treatment, 100 µM histamine and 10 µg/ml histamine-loaded poly (lactic-co-glycolic acid) microparticles significantly stimulated microglia motility via H4R activation. In addition, migration involves α5ß1 integrins, and p38 and Akt signaling pathways. Migration of microglial cells was also enhanced in the presence of lipopolysaccharide (LPS, 100 ng/ml), used as a positive control. Importantly, histamine inhibited LPS-stimulated migration via H4R activation. Histamine or H4R agonist also inhibited LPS-induced IL-1ß release in both N9 microglia cell line and hippocampal organotypic slice cultures. CONCLUSIONS: To our knowledge, we are the first to show a dual role of histamine in the modulation of microglial inflammatory responses. Altogether, our data suggest that histamine per se triggers microglia motility, whereas histamine impedes LPS-induced microglia migration and IL-1ß release. This last datum assigns a new putative anti-inflammatory role for histamine, acting via H4R to restrain exacerbated microglial responses under inflammatory challenge, which could have strong repercussions in the treatment of CNS disorders accompanied by microglia-derived inflammation.

Functional identification of neural stem cell-derived oligodendrocytes.

Directing neural stem cells (NSCs) differentiation towards oligodendroglial cell lineage is a crucial step in the endeavor of developing cell replacement-based therapies for demyelinating diseases. Evaluation of NSCs differentiation is mostly performed by methodologies that use fixed cells, like immunocytochemistry, or lysates, like Western blot. On the other hand, electrophysiology allows differentiation studies on living cells, but it is highly time-consuming and endowed with important limitations concerning population studies. Herein, we describe a functional method, based on single cell calcium imaging, which accurately and rapidly distinguishes cell types among NSCs progeny, in living cultures prepared from the major reservoir of NSCs in the postnatal mouse brain, the subventricular zone (SVZ). Indeed, by applying a rational sequence of three stimuli-KCl, histamine, and thrombin-to the heterogeneous SVZ cell population, one can identify each cell phenotype according to its unique calcium signature. Mature oligodendrocytes, the myelin-forming cells of the central nervous system, are the thrombin-responsive cells in SVZ cell culture and display no intracellular calcium increase upon KCl or histamine perfusion. On the other hand, KCl and histamine stimulate neurons and immature cells, respectively. The method described in this chapter is a valuable tool to identify novel pro-oligodendrogenic compounds, which may play an important role in the design of future treatments for demyelinating disorders such as multiple sclerosis.

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.

Neuropeptide Y promotes neurogenesis and protection against methamphetamine-induced toxicity in mouse dentate gyrus-derived neurosphere cultures.

Methamphetamine (METH) is a psychostimulant drug of abuse that causes severe brain damage. However, the mechanisms responsible for these effects are poorly understood, particularly regarding the impact of METH on hippocampal neurogenesis. Moreover, neuropeptide Y (NPY) is known to be neuroprotective under several pathological conditions. Here, we investigated the effect of METH on dentate gyrus (DG) neurogenesis, regarding cell death, proliferation and differentiation, as well as the role of NPY by itself and against METH-induced toxicity. DG-derived neurosphere cultures were used to evaluate the effect of METH or NPY on cell death, proliferation or neuronal differentiation. Moreover, the role of NPY and its receptors (Y(1), Y(2) and Y(5)) was investigated under conditions of METH-induced DG cell death. METH-induced cell death by both apoptosis and necrosis at concentrations above 10 nM, without affecting cell proliferation. Furthermore, at a non-toxic concentration (1 nM), METH decreased neuronal differentiation. NPY's protective effect was mainly due to the reduction of glutamate release, and it also increased DG cell proliferation and neuronal differentiation via Y(1) receptors. In addition, while the activation of Y(1) or Y(2) receptors was able to prevent METH-induced cell death, the Y(1) subtype alone was responsible for blocking the decrease in neuronal differentiation induced by the drug. Taken together, METH negatively affects DG cell viability and neurogenesis, and NPY is revealed to be a promising protective tool against the deleterious effects of METH on hippocampal neurogenesis.

Neuropeptide Y inhibits interleukin-1 beta-induced microglia motility.

Increasing evidences suggest that neuropeptide Y (NPY) may act as a key modulator of the cross-talk between the brain and the immune system in health and disease. In the present study, we dissected the possible inhibitory role of NPY upon inflammation-associated microglial cell motility. NPY, through activation of Y(1) receptors, was found to inhibit lipopolysaccharide (LPS)-induced microglia (N9 cell line) motility. Moreover, stimulation of microglia with LPS was inhibited by IL-1 receptor antagonist (IL-1ra), suggesting the involvement of endogenous interleukin-1 beta (IL-1ß) in this process. Direct stimulation with IL-1ß promoted downstream p38 mitogen-activated protein kinase mobilization and increased microglia motility. Moreover, consistently, p38 mitogen-activated protein kinase inhibition decreased the extent of actin filament reorganization occurring during plasma membrane ruffling and p38 phosphorylation was inhibited by NPY, involving Y(1) receptors. Significantly, the key inhibitory role of NPY on LPS-induced motility of CD11b-positive cells was further confirmed in mouse brain cortex explants. In summary, we revealed a novel functional role for NPY in the regulation of microglial function that may have important implications in the modulation of CNS injuries/diseases where microglia migration/motility might play a role.

Functional evaluation of neural stem cell differentiation by single cell calcium imaging.

Neurogenesis in the adult mammalian brain occurs in two specific brain areas, the subventricular zone (SVZ) bordering the lateral ventricles and the subgranular zone (SGZ) of the hippocampus. Although these regions are prone to produce new neurons, cultured cells from these neurogenic niches tend to be mixed cultures, containing both neurons and glial cells. Several reports highlight the potential of the self-healing capacity of the brain following injury. Even though much knowledge has been produced on the neurogenesis itself, brain repairing strategies are still far away from patients cure. Here we review general concepts in the neurogenesis field, also addressing the methods available to study neural stem cell differentiation. A major problem faced by research groups and companies dedicated to brain regenerative medicine resides on the lack of good methods to functionally identify neural stem cell differentiation and novel drug targets. To address this issue, we developed a unique single cell calcium imaging-based method to functionally discriminate different cell types derived from SVZ neural stem cell cultures. The unique functional profile of each SVZ cell type was correlated at the single cell level with the immunodetection of specific phenotypic markers. This platform was raised on the basis of the functional response of neurons, oligodendrocytes and immature cells to depolarising agents, to thrombin and to histamine, respectively. We also outline key studies in which our new platform was extremely relevant in the context of drug discovery and development in the area of brain regenerative medicine.