Astrocyte Migration Research Articles

The extracellular matrix molecule tenascin C modulates expression levels and territories of key patterning genes during spinal cord astrocyte specification

The generation of astrocytes during the development of the mammalian spinal cord is poorly understood. Here, we demonstrate for the first time that the extracellular matrix glycoprotein tenascin C regulates the expression of key patterning genes during late embryonic spinal cord development, leading to a timely maturation of gliogenic neural precursor cells. We first show that tenascin C is expressed by gliogenic neural precursor cells during late embryonic development. The loss of tenascin C leads to a sustained generation and delayed migration of Fgfr3-expressing immature astrocytes in vivo. Consistent with an increased generation of astroglial cells, we documented an increased number of GFAP-positive astrocytes at later stages. Mechanistically, we could demonstrate an upregulation and domain shift of the patterning genes Nkx6.1 and Nkx2.2 in vivo. In addition, sulfatase 1, a known downstream target of Nkx2.2 in the ventral spinal cord, was also upregulated. Sulfatase 1 regulates growth factor signalling by cleaving sulphate residues from heparan sulphate proteoglycans. Consistent with this function, we observed changes in both FGF2 and EGF responsiveness of spinal cord neural precursor cells. Taken together, our data implicate Tnc in the regulation of proliferation and lineage progression of astroglial progenitors in specific domains of the developing spinal cord.

Post‐Golgi Supramolecular Assembly of Aquaporin‐4 in Orthogonal Arrays

The supramolecular assembly of aquaporin-4 (AQP4) in orthogonal arrays of particles (OAPs) involves N-terminus interactions of the M23-AQP4 isoform. We found AQP4 OAPs in cell plasma membranes but not in endoplasmic reticulum (ER) or Golgi, as shown by: (i) native gel electrophoresis of brain and AQP4-transfected cells, (ii) photobleaching recovery of green fluorescent protein-AQP4 chimeras in live cells and (iii) freeze-fracture electron microscopy (FFEM). We found that AQP4 OAP formation in plasma membranes, but not in the Golgi, was not related to AQP4 density, pH, membrane lipid composition, C-terminal PDZ domain interactions or α-syntrophin expression. Remarkably, however, fusion of AQP4-containing Golgi vesicles with (AQP4-free) plasma membrane vesicles produced OAPs, suggesting the involvement of plasma membrane factor(s) in AQP4 OAP formation. In investigating additional possible determinants of OAP assembly we discovered membrane curvature-dependent OAP assembly, in which OAPs were disrupted by extrusion of plasma membrane vesicles to ∼110 nm diameter, but not to ∼220 nm diameter. We conclude that AQP4 supramolecular assembly in OAPs is a post-Golgi phenomenon involving plasma membrane-specific factor(s). Post-Golgi and membrane curvature-dependent OAP assembly may be important for vesicle transport of AQP4 in the secretory pathway and AQP4-facilitated astrocyte migration, and suggests a novel therapeutic approach for neuromyelitis optica.

Role of Connective Tissue Growth Factor in the Retinal Vasculature during Development and Ischemia

To investigate the function of connective tissue growth factor (CTGF), a matricellular protein of the CCN (Cyr61/CTGF/Nov) family, in retinal vasculature during development and ischemia. CTGF expression was determined using RT-PCR, immunohistochemistry, and transgenic mice carrying CTGF promoter-driven-GFP. CTGF antibody was intraocularly injected into neonates at postnatal day (P)2, and its effect on retinal angiogenesis was analyzed at P4. Transgenic animals expressing GFP regulated by the glial fibrillary acidic protein promoter were used for astrocyte visualization. Retinal vascular occlusion was introduced by rose Bengal and laser photocoagulation on chimeric mice that were reconstituted with GFP+ bone marrow cells. Vascular repair in response to VEGF-A and CTGF was analyzed. A temporal increase in CTGF at both mRNA and protein levels was observed in the ganglion cell layer and inner nuclear layer during development. Endothelial cells and pericytes were identified as the main cellular sources of CTGF during retinal angiogenesis. CTGF stimulated the migration of astrocytes, retinal endothelial cells, and pericytes in vitro. Inhibition of CTGF by specific antibody affected vascular filopodial extension, growth of the superficial vascular plexus, and astrocyte remodeling. In adult mice, CTGF was prominently expressed in the perivascular cells of arteries. CTGF activated bone marrow-derived perivascular cells and promoted fibrovascular membrane formation in the laser-induced adult retinopathy model. CTGF is expressed in vascular beds and acts on multiple cell types. It is important for vessel growth during early retinal development and promotes the fibrovascular reaction in murine retinal ischemia after laser injury.

A Complex of Nuclear Factor I-X3 and STAT3 Regulates Astrocyte and Glioma Migration through the Secreted Glycoprotein YKL-40

Nuclear factor I-X3 (NFI-X3) is a newly identified splice variant of NFI-X that regulates expression of several astrocyte-specific markers, such as glial fibrillary acidic protein. Here, we identified a set of genes regulated by NFI-X3 that includes a gene encoding a secreted glycoprotein YKL-40. Although YKL-40 expression is up-regulated in glioblastoma multiforme, its regulation and functions in nontransformed cells of the central nervous system are widely unexplored. We find that expression of YKL-40 is activated during brain development and also differentiation of neural progenitors into astrocytes in vitro. Furthermore, YKL-40 is a migration factor for primary astrocytes, and its expression is controlled by both NFI-X3 and STAT3, which are known regulators of gliogenesis. Knockdown of NFI-X3 and STAT3 significantly reduced YKL-40 expression in astrocytes, whereas overexpression of NFI-X3 dramatically enhanced YKL-40 expression in glioma cells. Activation of STAT3 by oncostatin M induced YKL-40 expression in astrocytes, whereas expression of a dominant-negative STAT3 had a suppressive effect. Mechanistically, NFI-X3 and STAT3 form a complex that binds to weak regulatory elements in the YKL-40 promoter and activates transcription. We propose that NFI-X3 and STAT3 control the migration of differentiating astrocytes as well as migration and invasion of glioma cells via regulating YKL-40 expression.

Beneficial compaction of spinal cord lesion by migrating astrocytes through glycogen synthase kinase‐3 inhibition

The migratory response of astrocytes is essential for restricting inflammation and preserving tissue function after spinal cord injury (SCI), but the mechanisms involved are poorly understood. Here, we observed stimulation of in vitro astrocyte migration by the new potent glycogen synthase kinase-3 (GSK-3) inhibitor Ro3303544 and investigated the effect of Ro3303544 administration for 5 days following SCI in mice. This treatment resulted in accelerated migration of reactive astrocytes to sequester inflammatory cells that spared myelinated fibres and significantly promoted functional recovery. Moreover, the decreased extent of chondroitin sulphate proteoglycans and collagen IV demonstrated that scarring was reduced in Ro3303544-treated mice. A variety of in vitro and in vivo experiments further suggested that GSK-3 inhibition stimulated astrocyte migration by decreasing adhesive activity via reduced surface expression of β1-integrin. Our results reveal a novel benefit of GSK-3 inhibition for SCI and suggest that the stimulation of astrocyte migration is a feasible therapeutic strategy for traumatic injury in the central nervous system.

Astrocyte-Derived Tissue Transglutaminase Interacts with Fibronectin: A Role in Astrocyte Adhesion and Migration?

An important neuropathological feature of neuroinflammatory processes that occur during e.g. Multiple Sclerosis (MS) is the formation of an astroglial scar. Astroglial scar formation is facilitated by the interaction between astrocytes and extracellular matrix proteins (ECM) such as fibronectin. Since there is evidence indicating that glial scars strongly inhibit both axon growth and (re)myelination in brain lesions, it is important to understand the factors that contribute to the interaction between astrocytes and ECM proteins. Tissue Transglutaminase (TG2) is a multifunctional enzyme with an ubiquitous tissue distribution, being clearly present within the brain. It has been shown that inflammatory cytokines can enhance TG2 activity. In addition, TG2 can mediate cell adhesion and migration and it binds fibronectin with high affinity. We therefore hypothesized that TG2 is involved in astrocyte-fibronectin interactions. Our studies using primary rat astrocytes show that intracellular and cell surface expression and activity of TG2 is increased after treatment with pro-inflammatory cytokines. Astrocyte-derived TG2 interacts with fibronectin and is involved in astrocyte adhesion onto and migration across fibronectin. TG2 is involved in stimulating focal adhesion formation which is necessary for the interaction of astrocytes with ECM proteins. We conclude that astrocyte-derived TG2 contributes to the interaction between astrocytes and fibronectin. It might thereby regulate ECM remodeling and possibly glial scarring.

Abstract A67: Transcription factor IRF3 inhibits glioma cell invasiveness

Abstract Malignant gliomas are the most common primary brain tumors with a high rate of mortality. The major problem for successful cure of gliomas is the ability of tumor cells to infiltrate into surrounding brain tissue. The prevention of infiltration will allow localizing the tumor following surgical dissection. A number of different approaches have been used to inhibit glioblastoma cell invasiveness. In the present work, we have tested the possibility that the transcription factor interferon regulatory factor 3 (IRF3) gene transfer could inhibit glioma invasiveness. IRF3 is a known component of TLR3 and TLR4 signaling which is required for IFNβ transcription and antiviral immunity. We used glioblastoma cell lines U251 and U87 as in vitro model systems. Tumor cells were transfected with an adenovirus construct bearing IRF3 (Ad-IRF3) or Ad-GFP as a control and were subsequently treated with a combination of cytokines (IL-1 β and IFNγ) to mimic the inflammatory tumor environment. Parallel experiments were performed with primary human fetal astrocytes. Subsequent analyses included the wound healing assay, BD Matrigel invasion assay, real-time PCR, microarray, Western blotting, and ELISA. First, we observed inhibition of astrocyte migration by IRF3, especially when the cells were exposed to cytokines. Then we found that IRF3 significantly inhibits the migration and invasion of the glioma cells. Real-time PCR with primary astrocytes and glioma cells indicated that IRF3 inhibited the expression of a number metalloproteases including MMP-7 and MMP-9. Additionally, we examined the expression of cytokines and chemokines which participate in the brain innate immune response and neuroinflammatory responses to tumors. In primary astrocytes, we found unique ability of IRF3 to regulate glial immune responses, i.e., upregulation of anti-inflammatory or regulatory cytokine genes (IFNβ, IL-27, and IL-13) and downregulation of proinflammatory genes (iNOS, CXCL1, IL-8, and TNFα). A similar picture was observed in glioma cells as well, including the production of antiangiogenic and antiproliferative cytokine, IFNβ. Interestingly, we found that glioma cells produced robust amounts of IL-1β in response to cytokine treatment, whereas normal human astrocytes produced none, and this was significantly inhibited by IRF3. As IL-1β is a neurotoxin and is a major proinflammatory cytokine that activates MMPs and other inflammatory mediators, these results have implications for the pathogenesis and therapy of malignant gliomas. Our data implicate IL-1 as a potential mediator of glioma invasiveness and neurotoxicity and IRF3 as a potential therapy target for malignant gliomas. Acknowledgement: This work was supported by the Einstein Molecular Neuropathology Training Grant T32NS007098, NIH RO1 MH55477, and the Einstein Human Fetal Tissue Repository. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr A67.

Integrin-dependent and -independent functions of astrocytic fibronectin in retinal angiogenesis

Fibronectin (FN) is a major component of the extracellular matrix and functions in cell adhesion, cell spreading and cell migration. In the retina, FN is transiently expressed and assembled on astrocytes (ACs), which guide sprouting tip cells and deposit a provisional matrix for sprouting angiogenesis. The precise function of FN in retinal angiogenesis is largely unknown. Using genetic tools, we show that astrocytes are the major source of cellular FN during angiogenesis in the mouse retina. Deletion of astrocytic FN reduces radial endothelial migration during vascular plexus formation in a gene dose-dependent manner. This effect correlates with reduced VEGF receptor 2 and PI3K/AKT signalling, and can be mimicked by selectively inhibiting VEGF-A binding to FN through intraocular injection of blocking peptides. By contrast, AC-specific replacement of the integrin-binding RGD sequence with FN-RGE or endothelial deletion of itga5 shows little effect on migration and PI3K/AKT signalling, but impairs filopodial alignment along AC processes, suggesting that FN-integrin α5β1 interaction is involved in filopodial adhesion to the astrocytic matrix. AC FN shares its VEGF-binding function and cell-surface distribution with heparan-sulfate (HS), and genetic deletion of both FN and HS together greatly enhances the migration defect, indicating a synergistic function of FN and HS in VEGF binding. We propose that in vivo the VEGF-binding properties of FN and HS promote directional tip cell migration, whereas FN integrin-binding functions to support filopodia adhesion to the astrocytic migration template.

Sphingosine Kinase 1 and Sphingosine 1-Phosphate Receptor 3 Are Functionally Upregulated on Astrocytes under Pro-Inflammatory Conditions

BackgroundReactive astrocytes are implicated in the development and maintenance of neuroinflammation in the demyelinating disease multiple sclerosis (MS). The sphingosine kinase 1 (SphK1)/sphingosine1-phosphate (S1P) receptor signaling pathway is involved in modulation of the inflammatory response in many cell types, but the role of S1P receptor subtype 3 (S1P3) signaling and SphK1 in activated rat astrocytes has not been defined.Methodology/Principal FindingsUsing immunohistochemistry we observed the upregulation of S1P3 and SphK1 expression on reactive astrocytes and SphK1 on macrophages in MS lesions. Increased mRNA and protein expression of S1P3 and SphK1, as measured by qPCR and Western blotting respectively, was observed after treatment of rat primary astrocyte cultures with the pro-inflammatory stimulus lipopolysaccharide (LPS). Activation of SphK by LPS stimulation was confirmed by SphK activity assay and was blocked by the use of the SphK inhibitor SKI (2-(p-hydroxyanilino)-4-(p-chlorphenyl) thiazole. Treatment of astrocytes with a selective S1P3 agonist led to increased phosphorylation of extracellular signal-regulated kinase (ERK)-1/2), which was further elevated with a LPS pre-challenge, suggesting that S1P3 upregulation can lead to increased functionality. Moreover, astrocyte migration in a scratch assay was induced by S1P and LPS and this LPS-induced migration was sensitive to inhibition of SphK1, and independent of cell proliferation. In addition, S1P induced secretion of the potentially neuroprotective chemokine CXCL1, which was increased when astrocytes were pre-challenged with LPS. A more prominent role of S1P3 signaling compared to S1P1 signaling was demonstrated by the use of selective S1P3 or S1P1 agonists.Conclusion/SignificanceIn summary, our data demonstrate that the SphK1/S1P3 signaling axis is upregulated when astrocytes are activated by LPS. This signaling pathway appears to play a role in the establishment and maintenance of astrocyte activation. Upregulation of the pathway in MS may be detrimental, e.g. through enhancing astrogliosis, or beneficial through increased remyelination via CXCL1.

Nitric oxide production by endothelin‐1 enhances astrocytic migration via the tyrosine nitration of matrix metalloproteinase‐9

The deleterious effects of endothelin-1 (ET-1) in the central nervous system (CNS) include disturbance of water homeostasis and blood-brain barrier (BBB) integrity. In the CNS, ischemic injury elicits ET-1 release from astrocytes, behaving through G-protein coupled ET receptors. These considerations raise the question of whether ET-1 influences cellular functions of astrocytes, the major cell type that provides structural and functional support for neurons. Uncontrolled nitric oxide (NO) production has been implicated in sterile brain insults, neuroinflammation, and neurodegenerative diseases, which involve astrocyte activation and neuronal death. However, the detailed mechanisms of ET-1 action related to NO release on rat brain astrocytes (RBA-1) remain unknown. In this study, we demonstrate that exposure of astrocytes to ET-1 results in the inducible nitric oxide synthase (iNOS) up-regulation, NO production, and matrix metalloproteinase-9 (MMP-9) activation in astrocytes. The data obtained with Western blot, reverse transcription-PCR (RT-PCR), and immunofluorescent staining analyses showed that ET-1-induced iNOS expression and NO production were mediated through an ET(B)-dependent transcriptional activation. Engagement of G(i/o)--and G(q) -coupled ET(B) receptors by ET-1 led to activation of c-Src-dependent phosphoinositide 3-kinase (PI3K)/Akt and p42/p44 mitogen-activated protein kinase (MAPK) and then activated transcription factor nuclear factor-κB (NF-κB). The activated NF-κB was translocated into nucleus and thereby promoted iNOS gene transcription. Ultimately, NO production stimulated by ET-1 enhanced the migration of astrocytes through the tyrosine nitration of MMP-9. Taken together, these results suggested that in astrocytes, activation of NF-κB by ET(B)-dependent c-Src, PI3K/Akt, and p42/p44 MAPK signalings is necessary for ET-1-induced iNOS gene up-regulation.

CPEB1 modulates lipopolysaccharide-mediated iNOS induction in rat primary astrocytes

Upon CNS damage, astrocytes undergo a series of biological changes including increased proliferation, production of inflammatory mediators and morphological changes, in a response collectively called reactive gliosis. This process is an essential part of the brains response to injury, yet much is unknown about the molecular mechanism(s) that induce these changes. In this study, we investigated the role of cytoplasmic polyadenylation element binding protein 1 (CPEB1) in the regulation of inflammatory responses in a model of reactive gliosis, lipopolysaccharide-stimulated astrocytes. CPEB1 is an mRNA-binding protein recently shown to be expressed in astrocytes that may play a role in astrocytes migration. After LPS stimulation, the expression and phosphorylation of CPEB1 was increased in rat primary astrocytes in a JNK-dependent process. siRNA-induced knockdown of CPEB1 expression inhibited the LPS-induced up-regulation of iNOS as well as NO and ROS production, a hallmark of immunological activation of astrocytes. The results from the study suggest that CPEB1 is actively involved in the regulation of inflammatory responses in astrocytes, which might provide new insights into the regulatory mechanism after brain injury.

Aquaporin-4: orthogonal array assembly, CNS functions, and role in neuromyelitis optica

Aquaporin-4 (AQP4) is a water-selective transporter expressed in astrocytes throughout the central nervous system, as well as in kidney, lung, stomach and skeletal muscle. The two AQP4 isoforms produced by alternative spicing, M1 and M23 AQP4, form heterotetramers that assemble in cell plasma membranes in supramolecular structures called orthogonal arrays of particles (OAPs). Phenotype analysis of AQP4-null mice indicates the involvement of AQP4 in brain and spinal cord water balance, astrocyte migration, neural signal transduction and neuroinflammation. AQP4-null mice manifest reduced brain swelling in cytotoxic cerebral edema, but increased brain swelling in vasogenic edema and hydrocephalus. AQP4 deficiency also increases seizure duration, impairs glial scarring, and reduces the severity of autoimmune neuroinflammation. Each of these phenotypes is likely explicable on the basis of reduced astrocyte water permeability in AQP4 deficiency. AQP4 is also involved in the neuroinflammatory demyelinating disease neuromyelitis optica (NMO), where autoantibodies (NMO-IgG) targeting AQP4 produce astrocyte damage and inflammation. Mice administered NMO-IgG and human complement by intracerebral injection develop characteristic NMO lesions with neuroinflammation, demyelination, perivascular complement deposition and loss of glial fibrillary acidic protein and AQP4 immunoreactivity. Our findings suggest the potential utility of AQP4-based therapeutics, including small-molecule modulators of AQP4 water transport function for therapy of brain swelling, injury and epilepsy, as well as small-molecule or monoclonal antibody blockers of NMO-IgG binding to AQP4 for therapy of NMO.

Changes in Spinal Cord Expression of Fractalkine and its Receptor in a Rat Model of Disc Herniation by Autologous Nucleus Pulposus

Behavior, mRNA and immunohistochemical assessment of the expression of fractalkine (CX3CL1) and its receptor (CX3CR1) in a rat model of disc herniation by autologous nucleus pulposus (NP) implantation. To evaluate the changes in expression of fractalkine and its receptor in the spinal cord and their association with pain behavior in a rat model of disc herniation. Chemokines have recently been implicated in the pathophysiology of neuropathic pain. They mediate astrocytic migration and microglial proliferation, which are involved in the regulation of nociceptive transmission. Fractalkine is a chemokine, which participates in the mechanisms of neuropathic pain as a mediator of neuron-glia interactions. Sixty-six rats (NP-treated = 47, sham = 19) were implanted with autologous NP (approximately 3 mg) on the left L5 nerve root, just proximal to the dorsal root ganglion and tested for thermal hyperalgesia and mechanical allodynia before surgery and on days 1, 5, 10, 20, and 30 after surgery. The changes of expression of fractalkine and its receptor in the spinal cord were studied using real time PCR and immunohistochemistry. Rats developed ipsilateral mechanical allodynia at day 1 and bilateral thermal hyperalgesia at day 5 after surgery, and these changes in sensitivity persisted throughout the observation period. The expression of mRNA for fractalkine in the spinal cord was increased by day 5 and remained upregulated for the duration of the experiment. The immunostaining for fractalkine increased in neurons and astrocytes and that for the fractalkine receptor increased in microglia in the dorsal horn ipsilateral to the disc herniation. Our results indicate that autologous implantation of NP induces thermal hyperalgesia and mechanical allodynia, and leads to an upregulation of fractalkine and its receptor in spinal neurons and glia, implicating fractalkine in association with radicular pain.

A developmental defect in astrocytes inhibits programmed regression of the hyaloid vasculature in the mammalian eye

Previously we reported the novel observation that astrocytes ensheath the persistent hyaloid artery, both in the Nuc1 spontaneous mutant rat, and in human PFV (persistent fetal vasculature) disease (Developmental Dynamics 234:36–47, 2005). We now show that astrocytes isolated from both the optic nerve and retina of Nuc1 rats migrate faster than wild type astrocytes. Aquaporin 4 (AQP4), the major water channel in astrocytes, has been shown to be important in astrocyte migration. We demonstrate that AQP4 expression is elevated in the astrocytes in PFV conditions, and we hypothesize that this causes the cells to migrate abnormally into the vitreous where they ensheath the hyaloid artery. This abnormal association of astrocytes with the hyaloid artery may impede the normal macrophage-mediated remodeling and regression of the hyaloid system.

GDNF modifies reactive astrogliosis allowing robust axonal regeneration through Schwann cell-seeded guidance channels after spinal cord injury

Reactive astrogliosis impedes axonal regeneration after injuries to the mammalian central nervous system (CNS). Here we report that glial cell line-derived neurotrophic factor (GDNF), combined with transplanted Schwann cells (SCs), effectively reversed the inhibitory properties of astrocytes at graft–host interfaces allowing robust axonal regeneration, concomitant with vigorous migration of host astrocytes into SC-seeded semi-permeable guidance channels implanted into a right-sided spinal cord hemisection at the 10th thoracic (T10) level. Within the graft, migrated host astrocytes were in close association with regenerated axons. Astrocyte processes extended parallel to the axons, implying that the migrated astrocytes were not inhibitory and might have promoted directional growth of regenerated axons. In vitro, GDNF induced migration of SCs and astrocytes toward each other in an astrocyte–SC confrontation assay. GDNF also enhanced migration of astrocytes on a SC monolayer in an inverted coverslip migration assay, suggesting that this effect is mediated by direct cell–cell contact between the two cell types. Morphologically, GDNF administration reduced astrocyte hypertrophy and induced elongated process extension of these cells, similar to what was observed in vivo. Notably, GDNF treatment significantly reduced production of glial fibrillary acidic protein (GFAP) and chondroitin sulfate proteoglycans (CSPGs), two hallmarks of astrogliosis, in both the in vivo and in vitro models. Thus, our study demonstrates a novel role of GDNF in modifying spinal cord injury (SCI)-induced astrogliosis resulting in robust axonal regeneration in adult rats.

Analysis of Schwann-astrocyte Interactions Using <em>In Vitro</em> Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors 1,2 and providing growth promoting adhesion molecules 3 and extracellular matrix molecules 4. In addition they myelinate the demyelinated axons at the site of injury 5. However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes 6,7. This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules 8-13. In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour. These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries. The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer 14,15. This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip. Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) 16,17, FGF/Heparin 18, Eph/Ephrins19. This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.

Analysis of Schwann-astrocyte Interactions Using <em>In Vitro</em> Assays

Schwann cells are one of the commonly used cells in repair strategies following spinal cord injuries. Schwann cells are capable of supporting axonal regeneration and sprouting by secreting growth factors (1,2) and providing growth promoting adhesion molecules (3) and extracellular matrix molecules (4). In addition they myelinate the demyelinated axons at the site of injury (5). However following transplantation, Schwann cells do not migrate from the site of implant and do not intermingle with the host astrocytes (6,7). This results in formation of a sharp boundary between the Schwann cells and astrocytes, creating an obstacle for growing axons trying to exit the graft back into the host tissue proximally and distally. Astrocytes in contact with Schwann cells also undergo hypertrophy and up-regulate the inhibitory molecules (8-13). In vitro assays have been used to model Schwann cell-astrocyte interactions and have been important in understanding the mechanism underlying the cellular behaviour. These in vitro assays include boundary assay, where a co-culture is made using two different cells with each cell type occupying different territories with only a small gap separating the two cell fronts. As the cells divide and migrate, the two cellular fronts get closer to each other and finally collide. This allows the behaviour of the two cellular populations to be analyzed at the boundary. Another variation of the same technique is to mix the two cellular populations in culture and over time the two cell types segregate with Schwann cells clumped together as islands in between astrocytes together creating multiple Schwann-astrocyte boundaries. The second assay used in studying the interaction of two cell types is the migration assay where cellular movement can be tracked on the surface of the other cell type monolayer (14,15). This assay is commonly known as inverted coverslip assay. Schwann cells are cultured on small glass fragments and they are inverted face down onto the surface of astrocyte monolayers and migration is assessed from the edge of coverslip. Both assays have been instrumental in studying the underlying mechanisms involved in the cellular exclusion and boundary formation. Some of the molecules identified using these techniques include N-Cadherins 15, Chondroitin Sulphate proteoglycans(CSPGs) (16,17), FGF/Heparin (18), Eph/Ephrins(19). This article intends to describe boundary assay and migration assay in stepwise fashion and elucidate the possible technical problems that might occur.

Modulation of Glial and Neuronal Migration by Lipocalin-2 in Zebrafish

BackgroundGlial cells are involved in immune and inflammatory responses in the central nervous system (CNS). Glial cells such as microglia and astrocytes also provide structural and functional support for neurons. Migration and morphological changes of CNS cells are associated with their physiological as well as pathological functions. The secreted protein lipocalin-2 (LCN2) has been previously implicated in regulation of diverse cellular processes of glia and neurons, including cell migration and morphology.MethodsHere, we employed a zebrafish model to analyze the role of LCN2 in CNS cell migration and morphology in vivo. In the first part of this study, we examined the indirect effect of LCN2 on cell migration and morphology of microglia, astrocytes, and neurons cultured in vitro.ResultsConditioned media collected from LCN2-treated astrocytes augmented migration of glia and neurons in the Boyden chamber assay. The conditioned media also increased the number of neuronal processes. Next, in order to further understand the role of LCN2 in the CNS in vivo, LCN2 was ectopically expressed in the zebrafish spinal cord. Expression of exogenous LCN2 modulated neuronal cell migration in the spinal cord of zebrafish embryos, supporting the role of LCN2 as a cell migration regulator in the CNS.ConclusionThus, LCN2 proteins secreted under diverse conditions may play an important role in CNS immune and inflammatory responses by controlling cell migration and morphology.

Down-regulation of glutamine synthetase enhances migration of rat astrocytes after in vitro injury

Astrocytes undergo reactive transformation in response to physical injury (reactive gliosis) that may impede neural repair. Glutamine synthetase (GS) is highly expressed by astrocytes, and serves a neuroprotective function by converting cytotoxic glutamate and ammonia into glutamine. Glutamine synthetase was down-regulated in reactive astrocytes at the site of mechanical spinal cord injury (SCI) and in cultured astrocytes at the margins of a scratch wound, suggesting that GS may modulate reactive transformation and glial scar development. We evaluated this potential function of GS using siRNA-mediated GS knock-down. Suppression of astrocytic GS by GS siRNA increased cell migration into the scratch wound zone and decreased substrate adhesion as indicated by the number of focal adhesions expressing the adaptor protein paxillin. Migration was enhanced by glutamine and suppressed by glutamate, in contrast to the result expected if enhanced migration was due solely to changes in glutamine and glutamate concomitant with reduced GS activity. The membrane type 1-matrix metalloproteinase (MT1-MMP) was up-regulated in GS siRNA-treated astrocytes, while a broad-spectrum MMP antagonist inhibited migration in both wild type and GS knock-down astrocytes. In addition, GS siRNA inhibited expression of integrin β1, while antibody-mediated inhibition of integrin β1 impaired direction-specific protrusion and motility. Thus, GS may modulate motility and substrate adhesion through transmembrane integrin β1 signaling to the cytoskeleton and by MMT-mediated proteolysis of the extracellular matrix.

Transforming growth factor-β1 induces matrix metalloproteinase-9 and cell migration in astrocytes: roles of ROS-dependent ERK- and JNK-NF-κB pathways

BackgroundTransforming growth factor-β (TGF-β) and matrix metalloproteinases (MMPs) are the multifunctional factors during diverse physiological and pathological processes including development, wound healing, proliferation, and cancer metastasis. Both TGF-β and MMPs have been shown to play crucial roles in brain pathological changes. Thus, we investigated the molecular mechanisms underlying TGF-β1-induced MMP-9 expression in brain astrocytes.MethodsRat brain astrocytes (RBA-1) were used. MMP-9 expression was analyzed by gelatin zymography and RT-PCR. The involvement of signaling molecules including MAPKs and NF-κB in the responses was investigated using pharmacological inhibitors and dominant negative mutants, determined by western blot and gene promoter assay. The functional activity of MMP-9 was evaluated by cell migration assay.ResultsHere we report that TGF-β1 induces MMP-9 expression and enzymatic activity via a TGF-β receptor-activated reactive oxygen species (ROS)-dependent signaling pathway. ROS production leads to activation of extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun-N-terminal kinase (JNK) and then activation of the NF-κB transcription factor. Activated NF-κB turns on transcription of the MMP-9 gene. The rat MMP-9 promoter, containing a NF-κB cis-binding site, was identified as a crucial domain linking to TGF-β1 action.ConclusionsCollectively, in RBA-1 cells, activation of ERK1/2- and JNK-NF-κB cascades by a ROS-dependent manner is essential for MMP-9 up-regulation/activation and cell migration induced by TGF-β1. These findings indicate a new regulatory pathway of TGF-β1 in regulating expression of MMP-9 in brain astrocytes, which is involved in physiological and pathological tissue remodeling of central nervous system.