Fabienne Agasse

Tumor Necrosis Factor‐α Modulates Survival, Proliferation, and Neuronal Differentiation in Neonatal Subventricular Zone Cell Cultures

Tumor necrosis factor (TNF)‐α has been reported to modulate brain injury, but remarkably, little is known about its effects on neurogenesis. We report that TNF‐α strongly influences survival, proliferation, and neuronal differentiation in cultured subventricular zone (SVZ) neural stem/progenitor cells derived from the neonatal P1–3 C57BL/6 mice. By using single‐cell calcium imaging, we developed a method, based on cellular response to KCl and/or histamine, that allows the functional evaluation of neuronal differentiation. Exposure of SVZ cultures to 1 and 10 ng/ml mouse or 1 ng/ml human recombinant TNF‐α resulted in increased differentiation of cells displaying a neuronal‐like profile of [Ca2+]i responses, compared with the predominant profile of immature cells observed in control, nontreated cultures. Moreover, by using neutralizing antibodies for each TNF‐α receptor, we found that the proneurogenic effect of 1 ng/ml TNF‐α is mediated via tumor necrosis factor receptor 1 activation. Accordingly, the percentage of neuronal nuclear protein‐positive neurons was increased following exposure to mouse TNF‐α. Interestingly, exposure of SVZ cultures to 1 ng/ml TNF‐α induced cell proliferation, whereas 10 and 100 ng/ml TNF‐α induced apoptotic cell death. Moreover, we found that exposure of SVZ cells to TNF‐α for 15 minutes or 6 hours caused an increase in the phospho‐stress‐activated protein kinase/c‐Jun N‐terminal kinase immunoreactivity initially in the nucleus and then in growing axons, colocalizing with tau, consistent with axonogenesis. Taken together, these results show that TNF‐α induces neurogenesis in neonatal SVZ cell cultures of mice. TNF‐α, a proinflammatory cytokine and a proneurogenic factor, may play a central role in promoting neurogenesis and brain repair in response to brain injury and infection.

Neuropeptide Y Promotes Neurogenesis in Murine Subventricular Zone

Stem cells of the subventricular zone (SVZ) represent a reliable source of neurons for cell replacement. Neuropeptide Y (NPY) promotes neurogenesis in the hippocampal subgranular layer and the olfactory epithelium and may be useful for the stimulation of SVZ dynamic in brain repair purposes. We describe that NPY promotes SVZ neurogenesis. NPY (1 μM) treatments increased proliferation at 48 hours and neuronal differentiation at 7 days in SVZ cell cultures. NPY proneurogenic properties are mediated via the Y1 receptor. Accordingly, Y1 receptor is a major active NPY receptor in the mouse SVZ, as shown by functional autoradiography. Moreover, short exposure to NPY increased immunoreactivity for the phosphorylated form of extracellular signal‐regulated kinase 1/2 in the nucleus, compatible with a trigger for proliferation, whereas 6 hours of treatment amplified the phosphorylated form of c‐Jun‐NH2‐terminal kinase signal in growing axons, consistent with axonogenesis. NPY, as a promoter of SVZ neurogenesis, is a crucial factor for future development of cell‐based brain therapy.

Multifaces of neuropeptide Y in the brain – Neuroprotection, neurogenesis and neuroinflammation

Neuropeptide Y (NPY) has been implicated in the modulation of important features of neuronal physiology, including calcium homeostasis, neurotransmitter release and excitability. Moreover, NPY has been involved as an important modulator of hippocampal and thalamic circuits, receiving particular attention as an endogenous antiepileptic peptide and as a potential master regulator of feeding behavior. NPY not only inhibits excessive glutamate release (decreasing circuitry hyperexcitability) but also protects neurons from excitotoxic cell death. Furthermore, NPY has been involved in the modulation of the dynamics of dentate gyrus and subventricular zone neural stem cell niches. In both regions, NPY is part of the chemical resource of the neurogenic niche and acts through NPY Y1 receptors to promote neuronal differentiation. Interestingly, NPY is also considered a neuroimmune messenger. In this review, we highlight recent evidences concerning paracrine/autocrine actions of NPY involved in neuroprotection, neurogenesis and neuroinflammation. In summary, the three faces of NPY, discussed in the present review, may contribute to better understand the dynamics and cell fate decision in the brain parenchyma and in restricted areas of neurogenic niches, in health and disease.

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.

Nitric oxide stimulates the proliferation of neural stem cells bypassing the epidermal growth factor receptor.

Nitric oxide (NO) was described to inhibit the proliferation of neural stem cells. Some evidence suggests that NO, under certain conditions, can also promote cell proliferation, although the mechanisms responsible for a potential proliferative effect of NO in neural stem cells have remained unaddressed. In this work, we investigated and characterized the proliferative effect of NO in cell cultures obtained from the mouse subventricular zone. We found that the NO donor NOC‐18 (10 μM) increased cell proliferation, whereas higher concentrations (100 μM) inhibited cell proliferation. Increased cell proliferation was detected rapidly following exposure to NO and was prevented by blocking the mitogen‐activated kinase (MAPK) pathway, independently of the epidermal growth factor (EGF) receptor. Downstream of the EGF receptor, NO activated p21Ras and the MAPK pathway, resulting in a decrease in the nuclear presence of the cyclin‐dependent kinase inhibitor 1, p27KIP1, allowing for cell cycle progression. Furthermore, in a mouse model that shows increased proliferation of neural stem cells in the hippocampus following seizure injury, we observed that the absence of inducible nitric oxide synthase (iNOS−/− mice) prevented the increase in cell proliferation observed following seizures in wild‐type mice, showing that NO from iNOS origin is important for increased cell proliferation following a brain insult. Overall, we show that NO is able to stimulate the proliferation of neural stem cells bypassing the EGF receptor and promoting cell division. Moreover, under pathophysiological conditions in vivo, NO from iNOS origin also promotes proliferation in the hippocampus. STEM CELLS 2010;28:1219–1230

Controlling the Neuronal Differentiation of Stem Cells by the Intracellular Delivery of Retinoic Acid-Loaded Nanoparticles

The manipulation of endogenous stem cell populations from the subventricular zone (SVZ), a neurogenic niche, creates an opportunity to induce neurogenesis and influence brain regenerative capacities in the adult brain. Herein, we demonstrate the ability of polyelectrolyte nanoparticles to induce neurogenesis exclusively after being internalized by SVZ stem cells. The nanoparticles are not cytotoxic for concentrations equal or below 10 μg/mL. The internalization process is rapid, and nanoparticles escape endosomal fate in a few hours. Retinoic acid-loaded nanoparticles increase the number of neuronal nuclear protein (NeuN)-positive neurons and functional neurons responding to depolarization with KCl and expressing NMDA receptor subunit type 1 (NR1). These nanoparticles offer an opportunity for in vivo delivery of proneurogenic factors and neurodegenerative disease treatment.

Endogenous hepatocyte growth factor is a niche signal for subventricular zone neural stem cell amplification and self-renewal.

Neural stem cells persist in the adult mammalian brain, within the subventricular zone (SVZ). The endogenous mechanisms underpinning SVZ neural stem cell proliferation, self‐renewal, and differentiation are not fully elucidated. In the present report, we describe a growth‐stimulatory activity of liver explant‐conditioned media on SVZ cell cultures and identify hepatocyte growth factor (HGF) as a major player in this effect. HGF exhibited a mitogenic activity on SVZ cell cultures in a mitogen‐activated protein kinase (MAPK) (ERK1/2)‐dependent manner as U0126, a specific MAPK inhibitor, blocked it. Combining a functional neurosphere forming assay with immunostaining for c‐Met, along with markers of SVZ cells subtypes, demonstrated that HGF promotes the expansion of neural stem‐like cells that form neurospheres and self‐renew. Immunostaining, HGF enzyme‐linked immunosorbent assay and Madin‐Darby canine kidney cell scattering assay indicated that SVZ cell cultures produce and release HGF. SVZ cell‐conditioned media induced proliferation on SVZ cell cultures, which was blocked by HGF‐neutralizing antibodies, hence implying that endogenously produced HGF accounts for a major part in SVZ mitogenic activity. Brain sections immunostaining revealed that HGF is produced by nestin‐expressing cells and c‐Met is expressed within the SVZ by immature cells. HGF intracerebroventricular injection promoted SVZ cell proliferation and increased the ability of these cells exposed in vivo to HGF to form neurospheres in vitro, whereas intracerebroventricular injection of HGF‐neutralizing antibodies decreased SVZ cell proliferation. The present study unravels a major role, both in vitro and in vivo, for endogenous HGF in SVZ neural stem cell growth and self‐renewal. STEM CELLS 2009;27:408–419

Neuropeptide Y as an Endogenous Antiepileptic, Neuroprotective and Pro-Neurogenic Peptide

Neuropeptide Y (NPY) is a small peptide important in cardiovascular physiology, feeding, anxiety, depression and epilepsy. In the hippocampus, NPY is mainly produced and released by GABAergic interneurons and inhibits glutamatergic neurotransmission in the excitatory tri-synaptic circuit. Under epileptic conditions, there is a robust overexpression of NPY and NPY receptors particularly in the granular and pyramidal cells, contributing to the tonic inhibition of glutamate release and consequently to control the spread of excitability into other brain structures. Recently, an important role was attributed to NPY in neuroprotection against excitotoxicity and in the modulation of neurogenesis. In the present review we discuss the potential relevance of NPY and NPY receptors in neuroprotection and neurogenesis, with implications for brain repair strategies. Recent patents describing new NPY receptor antagonists directed to treat obesity and cardiovascular disorders were published. However, the NPYergic system may also prove to be a good target for the treatment of pharmaco-resistant forms of temporal lobe epilepsy, by acting on hyperexcitability, neuronal death or brain repair. In order to achieve new NPY-based antiepileptic and brain repair strategies, selective NPY receptor agonists able to reach their targets in the epileptic brain must be developed in the near future.

Histamine modulates microglia function

BackgroundHistamine 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.MethodsFirstly, 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-1β) 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.ResultsAfter 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 α5β1 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-1β release in both N9 microglia cell line and hippocampal organotypic slice cultures.ConclusionsTo 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-1β 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.

The Angiogenic Factor Angiopoietin-1 Is a Proneurogenic Peptide on Subventricular Zone Stem/Progenitor Cells

In the adult mammalian brain, the subventricular zone (SVZ) hosts stem cells constantly generating new neurons. Angiopoietin-1 (Ang-1) is an endothelial growth factor with a critical role in division, survival, and adhesion of endothelial cells via Tie-2 receptor activity. Expression of Tie-2 in nonendothelial cells, especially neurons and stem cells, suggests that Ang-1 may be involved in neurogenesis. In the present work, we investigated the putative role of Ang-1 on SVZ neurogenesis. Immature cells from SVZ-derived neurospheres express Ang-1 and Tie-2 mRNA, suggesting a role for the Ang-1/Tie-2 system in the neurogenic niche. Moreover, we also found that Tie-2 protein expression is retained on differentiation in neurons and glial cells. Ang-1 triggered proliferation via activation of the ERK1/2 (extracellular signal-regulated kinase 1/2) mitogen-activated protein kinase (MAPK) kinase pathway but did not induce cell death. Accordingly, coincubation with an anti-Tie-2 neutralizing antibody prevented the pro-proliferative effect of Ang-1. Furthermore, Ang-1 increased the number of NeuN (neuronal nuclear protein)-positive neurons in cultures treated for 7 d, as well as the number of functional neurons, as assessed by monitoring [Ca2+]i rises after application of specific stimuli for neurons and immature cells. The proneurogenic effect of Ang-1 is mediated by Tie-2 activation and subsequent mTOR (mammalian target of rapamycin kinase) mobilization. In agreement, neuronal differentiation significantly decreased after exposure to an anti-Tie-2 neutralizing antibody and to rapamycin. Moreover, Ang-1 elicited the activation of the SAPK (stress-activated protein kinase)/JNK (c-Jun N-terminal kinase) MAPK, involved in axonogenesis. Our work shows a proneurogenic effect of Ang-1, highlighting the relevance of blood vessel/stem cell cross talk in health and disease.