MCP-1 induces migration of adult neural stem cells

As a model for brain inflammation we previously studied transcriptional profiles of tumor necrosis factor-alpha (TNF)treated U373 astroglioma cells. In previous work we were able to demonstrate that the chemokine monocyte chemoattractant protein-1 (MCP-1, SCYA2, CCL2, MCAF) expression in U373 cells was inducible by TNF-alpha treatment. Demonstrably MCP-1 mRNA and protein expression in U373 cells was sustainable over time and at the highest level of all genes analyzed (Schwamborn et al., BMC Genomics 4, 46, 2003). In the hematopoietic system MCP-1 is a CC chemokine that attracts monocytes, memory T lymphocytes, and natural killer cells. In search of further functions in brain inflammation we tested the hypothesis that MCP-1 acts as a chemokine on neural stem cells. Here we report that MCP-1 activates the migration capacity of rat-derived neural stem cells. The migration of stem cells in a Boyden chamber analysis was elevated after stimulation with MCP-1. Time-lapse video microscopy visualized the migration of single stem cells from neurospheres in MCP-1-treated cultures, whereas untreated cultures depicted no migration at all, but showed signs of sprouting. Expression of the MCP-1 receptor CCR2 in neurosphere cultures was verified by RT-PCR and immunofluorescence microscopy. Supernatants from TNF-treated U373 cells also induced migration of neural stem cells.

Migration and differentiation of neuronal precursors in the postnatal brain: insights from the subventricular zone and cerebellum

Fundação para a Ciência e a Tecnologia - SFRH/BD/42848/2008, através do Programa MIT_Portugal em Sistemas de Bioengenharia; projectos PTDC/SAUNEU/104415/2008 e Projecto ref. 96542 da Fundação Caloust Gulbenkian

The role of matrix metalloproteinase mmp-9 in peripheral neural system regeneration

Multiple lines of evidence show that matrix metalloproteinases (MMPs) are involved in the peripheral neural system degenerative and regenerative processes. MMP-9 was suggested in particular to play a role in the peripheral nerve after injury or during Wallerian degeneration. Interestingly, our previous analysis of Lpin1 mutant mice (which present morphological signs of active demyelination and acute inflammatory cell migration, similar to processes present in the PNS undergoing Wallerian degeneration) revealed an accumulation of MMP-9 in the endoneurium of affected animals. We therefore generated a mouse line lacking both the Lpin1 and the MMP-9 genes in order to determine if MMP-9 plays a role in either inhibition or potentiation of the demyelinating phenotype present in Lpin1 knockout mice. The inactivation of MMP-9 alone did not lead to defects in PNS structure or function. Interestingly we observed that the double mutant animals showed reduced nerve conduction velocity, lower myelin protein mRNA expressions, and had more histological abnormalities as compared to the Lpin1 single mutants. In addition, based on immunohistochemical analysis and macrophage markers mRNA expression, we found a lower macrophage content in the sciatic nerve of the double mutant animals. Together our data indicate that MMP-9 plays a role in macrophage recruitment during postinjury PNS regeneration processes and suggest that slower macrophage infiltration delays regenerative processes in PNS.

Reelin controls progenitor cell migration in the healty and pathological adult brain

Understanding the signals that control migration of neural progenitor cells in the adult brain may provide new therapeutic opportunities. Reelin is best known for its role in regulating cell migration during brain development, but we now demonstrate a novel function for reelin in the injured adult brain. First, we show that Reelin is upregulated around lesions. Second, experimentally increasing Reelin expression levels in healthy mouse brain leads to a change in the migratory behavior of subventricular zone-derived progenitors, triggering them to leave the rostral migratory stream (RMS) to which they are normally restricted during their migration to the olfactory bulb. Third, we reveal that Reelin increases endogenous progenitor cell dispersal in periventricular structures independently of any chemoattraction but via cell detachment and chemokinetic action, and thereby potentiates spontaneous cell recruitment to demyelination lesions in the corpus callosum. Conversely, animals lacking Reelin signaling exhibit reduced endogenous progenitor recruitment at the lesion site. Altogether, these results demonstrate that beyond its known role during brain development, Reelin is a key player in post-lesional cell migration in the adult brain. Finally our findings provide proof of concept that allowing progenitors to escape from the RMS is a potential therapeutic approach to promote myelin repair.

Myelin-associated proteins block the migration of olfactory ensheathing cells: an in vitro study using single cell tracking and traction force microscopy

Newly generated olfactory receptor axons grow from the peripheral to the central nervous system aided by olfactory ensheathing cells (OECs). Thus, OEC transplantation has emerged as a promising therapy for spinal cord injuries and for other neural diseases. However, these cells do not present a uniform population, but, instead, a functionally heterogeneous population that exhibits a variety of responses including adhesion, repulsion and crossover during cell-cell and cell-matrix interactions. Some studies report that the migratory properties of OECs are compromised by inhibitory molecules and potentiated by chemical gradients. Here, we demonstrated that rodent OECs express all the components of the Nogo Receptor complex and that their migration is blocked by Myelin. Next, we used cell tracking and traction force microscopy to analyze OEC migration and its mechanical properties over Myelin. Our data relate the absence of traction force of OEC with lower migratory capacity, which correlates with changes in the F-Actin cytoskeleton and focal adhesion distribution. Lastly, OEC traction force and migratory capacity is enhanced after cell incubation with the Nogo Receptor inhibitor NEP1-40.

Tangential migration of neuronal precursors of glutamatergic neurons in the adult mammalian brain.

In a classic model of mammalian brain formation, precursors of principal glutamatergic neurons migrate radially along radial glia fibers whereas GABAergic interneuron precursors migrate tangentially. These migration modes have significant implications for brain function. Here we used clonal lineage tracing of active radial glia-like neural stem cells in the adult mouse dentate gyrus and made the surprising discovery that proliferating neuronal precursors of glutamatergic granule neurons exhibit significant tangential migration along blood vessels, followed by limited radial migration. Genetic birthdating and morphological and molecular analyses pinpointed the neuroblast stage as the main developmental window when tangential migration occurs. We also developed a partial "whole-mount" dentate gyrus preparation and observed a dense plexus of capillaries, with which only neuroblasts, among the entire population of progenitors, are directly associated. Together, these results provide insight into neuronal migration in the adult mammalian nervous system.

Potential role of NF-kappaB in adult neural stem cells: the underrated steersman?

Neural stem cells are precursors of neurons and glial cells. During brain development, these cells proliferate, migrate and differentiate into specific lineages. Recently neural stem cells within the adult central nervous system were identified. Informations are now emerging about regulation of stem cell proliferation, migration and differentiation by numerous soluble factors such as chemokines and cytokines. However, the signal transduction mechanisms downstream of these factors are less clear. Here, we review potential evidences for a novel central role of the transcription factor nuclear factor kappa B (NF-kappaB) in these crucial signal transduction processes. NF-kappaB is an inducible transcription factor detected in neurons, glia and neural stem cells. NF-kappaB was discovered by David Baltimore's laboratory as a transcription factor in lymphocytes. NF-kappaB is involved in many biological processes such as inflammation and innate immunity, development, apoptosis and anti-apoptosis. It has been recently shown that members of the NF-kappaB family are widely expressed by neurons, glia and neural stem cells. In the nervous system, NF-kappaB plays a crucial role in neuronal plasticity, learning, memory consolidation, neuroprotection and neurodegeneration. Recent data suggest an important role of NF-kappaB on proliferation, migration and differentiation of neural stem cells. NF-kappaB is composed of three subunits: two DNA-binding and one inhibitory subunit. Activation of NF-kappaB takes place in the cytoplasm and results in degradation of the inhibitory subunit, thus enabling the nuclear import of the DNA-binding subunits. Within the nucleus, several target genes could be activated. In this review, we suggest a model explaining the multiple action of NF-kappaB on neural stem cells. Furthermore, we discuss the potential role of NF-kappaB within the so-called brain cancer stem cells.

Tumor necrosis factor alpha triggers proliferation of adult neural stem cells via IKK/NF-kappaB signaling

BACKGROUND: Brain inflammation has been recognized as a complex phenomenon with numerous related aspects. In addition to the very well-described neurodegenerative effect of inflammation, several studies suggest that inflammatory signals exert a potentially positive influence on neural stem cell proliferation, migration and differentiation. Tumor necrosis factor alpha (TNF-alpha) is one of the best-characterized mediators of inflammation. To date, conclusions about the action of TNF on neural stem or progenitor cells (NSCs, NPCs) have been conflicting. TNF seems to activate NSC proliferation and to inhibit their differentiation into NPCs. The purpose of the present study was to analyze the molecular signal transduction mechanisms induced by TNF and resulting in NSC proliferation. RESULTS: Here we describe for the first time the TNF-mediated signal transduction cascade in neural stem cells (NSCs) that results in increased proliferation. Moreover, we demonstrate IKK-alpha/beta-dependent proliferation and markedly up-regulated cyclin D1 expression after TNF treatment. The significant increase in proliferation in TNF-treated cells was indicated by increased neurosphere volume, increased bromodeoxyuridin (BrdU) incorporation and a higher total cell number. Furthermore, TNF strongly activated nuclear factor-kappa B (NF-kappaB) as measured by reporter gene assays and by an activity-specific antibody. Proliferation of control and TNF-treated NSCs was strongly inhibited by expression of the NF-kappaB super-repressor IkappaB-AA1. Pharmacological blockade of IkappaB ubiquitin ligase activity led to comparable decreases in NF-kappaB activity and proliferation. In addition, IKK-beta gene product knock-down via siRNA led to diminished NF-kappaB activity, attenuated cyclin D1 expression and finally decreased proliferation. In contrast, TGFbeta-activated kinase 1 (TAK-1) is partially dispensable for TNF-mediated and endogenous proliferation. Understanding stem cell proliferation is crucial for future regenerative and anti-tumor medicine. CONCLUSION: TNF-mediated activation of IKK-beta resulted in activation of NF-kappaB and was followed by up-regulation of the bona-fide target gene cyclin D1. Activation of the canonical NF-kappaB pathway resulted in strongly increased proliferation of NSCs.

Neural stem cells, inflammation and NF-kappaB: basic principle of maintenance and repair or origin of brain tumours?

Several recent reports suggest that inflammatory signals play a decisive role in the self-renewal, migration and differentiation of multipotent neural stem cells (NSCs). NSCs are believed to be able to ameliorate the symptoms of several brain pathologies through proliferation, migration into the area of the lesion and either differentiation into the appropriate cell type or secretion of anti-inflammatory cytokines. Although NSCs have beneficial roles, current evidence indicates that brain tumours, such as astrogliomas or ependymomas are also caused by tumour-initiating cells with stem-like properties. However, little is known about the cellular and molecular processes potentially generating tumours from NSCs. Most pro-inflammatory conditions are considered to activate the transcription factor NF-kappaB in various cell types. Strong inductive effects of NF-kappaB on proliferation and migration of NSCs have been described. Moreover, NF-kappaB is constitutively active in most tumour cells described so far. Chronic inflammation is also known to initiate cancer. Thus, NF-kappaB might provide a novel mechanistic link between chronic inflammation, stem cells and cancer. This review discusses the apparently ambivalent role of NF-kappaB: physiological maintenance and repair of the brain via NSCs, and a potential role in tumour initiation. Furthermore, it reveals a possible mechanism of brain tumour formation based on inflammation and NF-kappaB activity in NSCs.

Administration of Neural Precursor Cells Ameliorates Renal Ischemia-Reperfusion Injury

In this study we evaluated whether administration of stem cells of neural origin (neural precursor cells, NPCs) could be protective against renal ischemia-reperfusion injury (IRI). We hypothesized that stem cell outcomes are not tissue-specific and that NPCs can improve tissue damage through paracrine mechanisms, especially due to immunomodulation. To this end, Wistar rats (200-250 g) were submitted to 1-hour ischemia and treated with NPCs (4 x 10(6) cells/animal) at 4 h of reperfusion. To serve as controls, ischemic animals were treated with cerebellum homogenate harvested from adult rat brain. All groups were sacrificed at 24 h of reperfusion. NPCs were isolated from rat fetus telencephalon and cultured until neurosphere formation (7 days). Before administration, NPCs were labeled with carboxyfluorescein diacetate succinimydylester (CFSE). Kidneys were harvested for analysis of cytokine profile and macrophage infiltration. At 24 h, NPC treatment resulted in a significant reduction in serum creatinine (IRI + NPC 1.21 + 0.18 vs. IRI 3.33 + 0.14 and IRI + cerebellum 2.95 + 0.78mg/dl, p < 0.05) and acute tubular necrosis (IRI + NPC 46.0 + 2.4% vs. IRI 79.7 + 14.2%, p < 0.05). NPC-CFSE and glial fibrillary acidic protein (GFAP)-positive cells (astrocyte marker) were found exclusively in renal parenchyma, which also presented GFAP and SOX-2 (an embryonic neural stem cell marker) mRNA expression. NPC treatment resulted in lower renal proinflammatory IL1-beta and TNF-alpha expression and higher anti-inflammatory IL-4 and IL-10 transcription. NPC-treated animals also had less macrophage infiltration and decreased serum proinflammatory cytokines (IL-1 beta, TNF-alpha and INF-gamma). Our data suggested that NPC therapy improved renal function by influencing immunological responses. Copyright (C) 2009 S. Karger AG, Basel

Extrinsic Purinergic Regulation of Neural Stem/Progenitor Cells: Implications for CNS Development and Repair

There has been tremendous progress in understanding neural stem cell (NSC) biology, with genetic and cell biological methods identifying sequential gene expression and molecular interactions guiding NSC specification into distinct neuronal and glial populations during development. Data has emerged on the possible exploitation of NSC-based strategies to repair adult diseased brain. However, despite increased information on lineage specific transcription factors, cell-cycle regulators and epigenetic factors involved in the fate and plasticity of NSCs, understanding of extracellular cues driving the behavior of embryonic and adult NSCs is still very limited. Knowledge of factors regulating brain development is crucial in understanding the pathogenetic mechanisms of brain dysfunction. Since injury-activated repair mechanisms in adult brain often recapitulate ontogenetic events, the identification of these players will also reveal novel regenerative strategies. Here, we highlight the purinergic system as a key emerging player in the endogenous control of NSCs. Purinergic signalling molecules (ATP, UTP and adenosine) act with growth factors in regulating the synchronized proliferation, migration, differentiation and death of NSCs during brain and spinal cord development. At early stages of development, transient and time-specific release of ATP is critical for initiating eye formation; once anatomical CNS structures are defined, purinergic molecules participate in calcium-dependent neuron-glia communication controlling NSC behaviour. When development is complete, some purinergic mechanisms are silenced, but can be re-activated in adult brain after injury, suggesting a role in regeneration and self-repair. Targeting the purinergic system to develop new strategies for neurodevelopmental disorders and neurodegenerative diseases will be also discussed.

Kinin-B2 Receptor Activity Determines the Differentiation Fate of Neural Stem Cells

Bradykinin is not only important for inflammation and blood pressure regulation, but also involved in neuromodulation and neuroprotection. Here we describe novel functions for bradykinin and the kinin-B2 receptor (B2BkR) in differentiation of neural stem cells. In the presence of the B2BkR antagonist HOE-140 during rat neurosphere differentiation, neuron-specific beta 3-tubulin and enolase expression was reduced together with an increase in glial protein expression, indicating that bradykinin- induced receptor activity contributes to neurogenesis. In agreement, HOE-140 affected in the same way expression levels of neural markers during neural differentiation of murine P19 and human iPS cells. Kinin-B1 receptor agonists and antagonists did not affect expression levels of neural markers, suggesting that bradykinin-mediated effects are exclusively mediated via B2BkR. Neurogenesis was augmented by bradykinin in the middle and late stages of the differentiation process. Chronic treatment with HOE-140 diminished eNOS and nNOS as well as M1-M4 muscarinic receptor expression and also affected purinergic receptor expression and activity. Neurogenesis, gliogenesis, and neural migration were altered during differentiation of neurospheres isolated from B2BkR knock-out mice. Whole mount in situ hybridization revealed the presence of B2BkR mRNA throughout the nervous system in mouse embryos, and less beta 3-tubulin and more glial proteins were expressed in developing and adult B2BkR knock-out mice brains. As a underlying transcriptional mechanism for neural fate determination, HOE-140 induced up-regulation of Notch1 and Stat3 gene expression. Because pharmacological treatments did not affect cell viability and proliferation, we conclude that bradykinin-induced signaling provides a switch for neural fate determination and specification of neurotransmitter receptor expression.