Astrocyte Migration Research Articles

Large is required for normal astrocyte migration and retinal vasculature development

BackgroundPersistent fetal vasculature (PFV) is a congenital developmental anomaly of the eye that accounts for about 5% of childhood blindness. The molecular mechanism of PFV remains unclear. As a glycosyltransferase of α-dystroglycan, LARGE mutations have been found in congenital muscular dystrophy patients with brain abnormalities. Spontaneous Large mutant mice displayed similar symptoms of human muscle–eye–brain disorders. However, the detailed roles of Large in ocular vasculature development still need to be uncovered.ResultsIn this paper, we report that a novel Large mutation generated by the piggyBac transposon insertion leads to PFV and abnormal retinal vasculature in mice. Glycosylation of α-DG, an essential component of the extracellular matrix, was significantly impaired in these Large mutants, leading to broken inner limiting membrane (ILM). As a guide of the retinal vasculature development, the distribution of retinal astrocytes became irregular within the retina, and many astrocytes abnormally migrated into the vitreous along with the hyaloid vessels in Large mutants.ConclusionsLarge is essential for ILM formation and retinal astrocyte migration. The novel Large mutant mouse can serve as a new PFV model to further dissect LARGE functions in ocular vasculature development.

Truncated TrkB.T1-Mediated Astrocyte Dysfunction Contributes to Impaired Motor Function and Neuropathic Pain after Spinal Cord Injury

Following spinal cord injury (SCI), astrocytes demonstrate long-lasting reactive changes, which are associated with the persistence of neuropathic pain and motor dysfunction. We previously demonstrated that upregulation of trkB.T1, a truncated isoform of the brain-derived neurotrophic factor receptor (BDNF), contributes to gliosis after SCI, but little is known about the effects of trkB.T1 on the function of astrocytes. As trkB.T1 is the sole isoform of trkB receptors expressed on astrocytes, we examined the function of trkB.T1-driven astrocytes in vitro and in vivo Immunohistochemistry showed that trkB.T1+ cells were significantly upregulated 7 d after injury, with sustained elevation in white matter through 8 weeks. The latter increase was predominantly found in astrocytes. TrkB.T1 was also highly expressed by neurons and microglia/macrophages at 7 d after injury and declined by 8 weeks. RNA sequencing of cultured astrocytes derived from trkB.T1+/+ (WT) and trkB.T1-/- (KO) mice revealed downregulation of migration and proliferation pathways in KO astrocytes. KO astrocytes also exhibited slower migration/proliferation in vitro in response to FBS or BDNF compared with WT astrocytes. Reduced proliferation of astrocytes was also confirmed after SCI in astrocyte-specific trkB.T1 KO mice; using mechanical allodynia and pain-related measurements on the CatWalk, these animals also showed reduced hyperpathic responses, along with improved motor coordination. Together, our data indicate that trkB.T1 in astrocytes contributes to neuropathic pain and neurological dysfunction following SCI, suggesting that trkB.T1 may provide a novel therapeutic target for SCI.SIGNIFICANCE STATEMENT Neuropathic pain after spinal cord injury (SCI) may in part be caused by upregulation of the brain-derived neurotrophic factor (BDNF) receptor trkB.T1, a truncated isoform of BDNF. TrkB.T1 is the only isoform of tropomyosin-related receptor kinase type B (trkB) receptors expressed on astrocytes. Here, we showed that trkB.T1 is significantly increased in the injured mouse spinal cord, where it is predominantly found in astrocytes. RNA sequencing of cultured astrocytes demonstrated downregulation of migration and proliferation pathways in trkB.T1 KO astrocytes. This was validated in vivo, where deletion of trkB.T1 in astrocytes reduced cell proliferation and migration. After SCI, astrocyte-specific trkB.T1 KO mice showed reduced hyperpathic responses and improved motor coordination. Therefore, the trkB.T1 receptor plays a significant pathophysiological role after SCI, and may provide a novel therapeutic target for SCI.

Fisetin regulates astrocyte migration and proliferation in vitro

Fisetin (3,3′,4′,7-tetrahydroxyflavone) is a plant flavonol found in fruits and vegetables that has been reported to inhibit migration and proliferation in several types of cancer. Reactive astrogliosis involves astrocyte migration and proliferation, and contributes to the formation of glial scars in central nervous system (CNS) disorders. However, the effect of fisetin on the migration and proliferation of astrocytes remains unclear. In this study, we found that fisetin inhibited astrocyte migration in a scratch-wound assay and diminished the phosphorylation of focal adhesion kinase (FAK; Tyr576/577 and paxillin (Tyr118). It also suppressed cell proliferation, as indicated by the decreased number of 5-ethynyl-2′-deoxyuridine (EdU)-positive cells, induced cell cycle arrest in the G1 phase, reduced the percentage of cells in the G2 and S phase (as measured by flow cytometry), and decreased cyclin D1 expression, but had no effect on apoptosis. Fisetin also decreased the phosphorylation levels of Akt and extracellular signal-regulated kinase (Erk)1/2, but had no effect on the phosphorylation of p38 mitogen-activated protein kinase (MAPK). These results indicate that fisetin inhibits aggressive cell phenotypes by suppressing cell migration and proliferation via the Akt/Erk signaling pathway. Fisetin may thus have potential for use as a therapeutic strategy targeting reactive astrocytes, which may lead to the inhibition of glial scar formation in vitro.

Issue Information

COVER PHOTOGRAPH: Fluorescence microscopy image of retinal flat‐mount stained with antibodies for PDGFRα, GFAP and PECAM‐1. From: A delay in vascularization induces abnormal astrocyte proliferation and migration in the mouse retina, Akane Morita, Hiroko Ushikubo, Asami Mori, Shiho Arima, Kenji Sakamoto, Tohru Nagamitsu, Kunio Ishii, and Tsutomu Nakahara; Developmental Dynamics 246:186–200.Read the full article at DOI: 10.1002/dvdy.24484

A delay in vascularization induces abnormal astrocyte proliferation and migration in the mouse retina.

Astrocytes migrate into the retina through the optic nerve head by means of the axons of retinal ganglion cells, and spread radially toward the peripheral retina. Endothelial cells migrate along the astrocyte cellular network to form the retinal surface vasculature. Here, we examined the effects of a delay in retinal vascularization on the migration and proliferation status of astrocytes in mice. A dose-dependent delay in retinal vascularization was observed in mice that had been treated with KRN633 (1-10 mg/kg), a VEGF receptor inhibitor, on the day of birth and on the following day. Delayed vascularization resulted in a delay in the astrocyte network formation, and an increase in astrocyte number in the optic nerve head and the vascular front. The increase in the number of astrocytes may be attributed to increased proliferation and delayed migration. These abnormalities in astrocyte behavior correlated with the degree of delay in retinal vascularization. The vascularization delay also led to retinal hypoxia, which subsequently stimulated VEGF leading to an increase in vascular density. These findings suggest that a delay in normal vascularization leads to abnormal astrocyte behavior, which results in the formation of abnormal astrocyte and endothelial cell networks in the mouse retina. Developmental Dynamics 246:186-200, 2017. © 2016 Wiley Periodicals, Inc.

Fidgetin regulates cultured astrocyte migration by severing tyrosinated microtubules at the leading edge.

Microtubule (MT) organization is essential for many cellular events, including mitosis, migration, and cell polarity. Fidgetin (Fign), an ATP-dependent, MT-severing protein, contributes to the regulation of MT configuration by cutting and trimming MT polymers. Functions of Fign have been indicated in neurite outgrowth, mitosis, meiosis, and cellular migration. Here we focus on migration of astrocytes. We find that Fign plays an essential role in cultured astrocyte migration by preferentially targeting MTs (or regions of MTs) that are rich in tyrosinated tubulin, a marker for especially dynamic MTs or especially dynamic regions of MTs. Inhibition of cellular migration induced by Fign knockdown can be rescued with concomitant knockdown of kinesin-12, a motor protein best known for its role in mitosis. We propose a novel working model for MT reconfiguration underlying cellular migration elicited by the functional cooperation of two distinct MT-related proteins.

Inhibition or ablation of transglutaminase 2 impairs astrocyte migration

Astrocytes play numerous complex roles that support and facilitate the function of neurons. Further, when there is an injury to the central nervous system (CNS) they can both facilitate or ameliorate functional recovery depending on the location and severity of the injury. When a CNS injury is relatively severe a glial scar is formed, which is primarily composed of astrocytes. The glial scar can be both beneficial, by limiting inflammation, and detrimental, by preventing neuronal projections, to functional recovery. Thus, understanding the processes and proteins that regulate astrocyte migration in response to injury is still of fundamental importance. One protein that is likely involved in astrocyte migration is transglutaminase 2 (TG2); a multifunctional protein expressed ubiquitously throughout the brain. Its functions include transamidation and GTPase activity, among others, and previous studies have implicated TG2 as a regulator of migration. Therefore, we examined the role of TG2 in primary astrocyte migration subsequent to injury. Using wild type or TG2−/− astrocytes, we manipulated the different functions and conformation of TG2 with novel irreversible inhibitors or mutant versions of the protein. Results showed that both inhibition and ablation of TG2 in primary astrocytes significantly inhibit migration. Additionally, we show that the deficiency in migration caused by deletion of TG2 can only be rescued with the native protein and not with mutants. Finally, the addition of TGFβ rescued the migration deficiency independent of TG2. Taken together, our study shows that transamidation and GTP/GDP-binding are necessary for inhibiting astrocyte migration and it is TGFβ independent.

Astrocytes in Migration.

Cell migration is a fundamental phenomenon that underlies tissue morphogenesis, wound healing, immune response, and cancer metastasis. Great progresses have been made in research methodologies, with cell migration identified as a highly orchestrated process. Brain is considered the most complex organ in the human body, containing many types of neural cells with astrocytes playing crucial roles in monitoring normal functions of the central nervous system. Astrocytes are mostly quiescent under normal physiological conditions in the adult brain but become migratory after injury. Under most known pathological conditions in the brain, spinal cord and retina, astrocytes are activated and become hypertrophic, hyperplastic, and up-regulating GFAP based on the grades of severity. These three observations are the hallmark in glia scar formation-astrogliosis. The reactivation process is initiated with structural changes involving cell process migration and ended with cell migration. Detailed mechanisms in astrocyte migration have not been studied extensively and remain largely unknown. Here, we therefore attempt to review the mechanisms in migration of astrocytes.

Retinal Proteoglycans Act as Cellular Receptors for Basement Membrane Assembly to Control Astrocyte Migration and Angiogenesis

The basement membrane is crucial for cell polarity, adhesion, and motility, but how it is assembled on the cell surface remains unclear. Here, we find that ablation of glycosaminoglycan (GAG) side chains of proteoglycans in the neuroretina disrupts the retinal basement membrane, leading to arrested astrocyte migration and reduced angiogenesis. Using genetic deletion and time-lapse imaging, we show that retinal astrocytes require neuronal-derived PDGF as a chemoattractive cue and the retinal basement membrane as a migratory substrate. Genetic ablation of heparan sulfates does not produce the same defects as GAG null mutants. In contrast, enzymatic removal of heparan sulfates and chondroitin sulfates together inhibits de novo laminin network assembly. These results indicate that both heparan and chondroitin sulfate proteoglycans participate in retinal basementmembrane assembly, thus promoting astrocyte migration and angiogenesis.

Relationship Between Obesity, Alzheimer's Disease, and Parkinson's Disease: an Astrocentric View.

Obesity is considered one of the greatest risk to human health and is associated with several factors including genetic components, diet, and physical inactivity. Recently, the relationship between obesity and numerous progressive and aging-related neurodegenerative diseases such as Parkinson's disease (PD) and Alzheimer's disease (AD) have been observed. Thus, the involvement of the most abundant and heterogeneous group of glial cells in neurodegenerative diseases, the astrocytes, is caused by a combination of the failure on their normal homeostatic functions and the increase of toxic metabolites upon pathological event. Upon brain damage, molecular signals induce astrocyte activation and migration to the site of injury, entering in a highly active state, with the aim to contribute to ameliorating or worsening the pathology. In this regard, the aim of this review is to elucidate the relationship between obesity, Alzheimer's disease, and Parkinson's disease and highlight the role of astrocytes in these pathologies.

Extracellular ATP induces graded reactive response of astrocytes and strengthens their antioxidative defense in vitro.

It is widely accepted that adenosine triphosphate (ATP) acts as a universal danger-associated molecular pattern with several known mechanisms for immune cell activation. In the central nervous system, ATP activates microglia and astrocytes and induces a neuroinflammatory response. The aim of the present study was to describe responses of isolated astrocytes to increasing concentrations of ATP (5 µM to 1 mM), which were intended to mimic graded intensity of the extracellular stimulus. The results show that ATP induces graded activation response of astrocytes in terms of the cell proliferation, stellation, shape remodeling, and underlying actin and GFAP filament rearrangement, although the changes occurred without an apparent increase in GFAP and actin protein expression. On the other hand, ATP in the range of applied concentrations did not evoke IL-1β release from cultured astrocytes, nor did it modify the release from LPS and LPS+IFN-γ-primed astrocytes. ATP did not promote astrocyte migration in the wound-healing assay, nor did it increase production of reactive oxygen and nitrogen species and lipid peroxidation. Instead, ATP strengthened the antioxidative defense of astrocytes by inducing Cu/ZnSOD and MnSOD activities and by increasing their glutathione content. Our current results suggest that although ATP triggers several attributes of activated astrocytic phenotype with a magnitude that increases with the concentration, it is not sufficient to induce full-blown reactive phenotype of astrocytes in vitro. © 2016 Wiley Periodicals, Inc.

Exposure to PM2.5 causes genetic changes in fetal rat cerebral cortex and hippocampus.

PM2.5 travels along the respiratory tract and enters systemic blood circulation. Studies have shown that PM2.5 increases the incidence of various diseases not only in adults but also in newborn infants. It causes chronic inflammation in pregnant women and retards fetal development. In this study, pregnant rats were exposed to PM2.5 for extended periods of time and it was found that PM2.5 exposure increased immune cells in mother rats. In addition, cytokines and free radicals rapidly accumulated in the amniotic fluid and indirectly affected the fetuses. The authors collected cerebral cortex and hippocampus samples at E18 and analyzed changes of miRNA levels. Expression levels of cortical miR-6315, miR-3588, and miR-466b-5p were upregulated, and positively correlated with the genes Pkn2 (astrocyte migration), Gorab (neuritogenesis), and Mobp (allergic encephalomyelitis). In contrast, PM2.5 decreased expression of miR-338-5p and let-7e-5p, both related to mental development. Further, PM2.5 exposure increased miR-3560 and let-7b-5p in the hippocampus, two proteins that regulate genes Oxct1 and Lin28b that control ketogenesis and glycosylation, and neural cell differentiation, respectively. miR-99b-5p, miR-92b-5p, and miR-99a-5p were decreased, leading to reduced expression of Kbtbd8 and Adam11 which reduced cell mitosis, migration, and differentiation, and inhibited learning abilities and motor coordination of the fetus. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1412-1425, 2017.

Depletion of kinesin-12, a myosin-IIB-interacting protein, promotes migration of cortical astrocytes

Kinesin-12 (also named Kif15) participates in important events during neuronal development, such as cell division of neuronal precursors, migration of young neurons and establishment of axons and dendritic arbors, by regulating microtubule organization. Little is known about the molecular mechanisms behind the functions of kinesin-12, and even less is known about its roles in other cell types of the nervous system. Here, we show that kinesin-12 depletion from cultured rat cortical astrocytes decreases cell proliferation but increases migration. Co-immunoprecipitation, GST pulldown and small interfering RNA (siRNA) experiments indicated that kinesin-12 directly interacts with myosin-IIB through their tail domains. Immunofluorescence analyses indicated that kinesin-12 and myosin-IIB colocalize in the lamellar region of astrocytes, and fluorescence resonance energy transfer analyses revealed an interaction between the two. The phosphorylation at Thr1142 of kinesin-12 was vital for their interaction. Loss of their interaction through expression of a phosphorylation mutant of kinesin-12 promoted astrocyte migration. We suggest that kinesin-12 and myosin-IIB can form a hetero-oligomer that generates force to integrate microtubules and actin filaments in certain regions of cells, and in the case of astrocytes, that this interaction can modulate their migration.

Selective Expression of Osteopontin in ALS-resistant Motor Neurons is a Critical Determinant of Late Phase Neurodegeneration Mediated by Matrix Metalloproteinase-9.

Differential vulnerability among motor neuron (MN) subtypes is a fundamental feature of amyotrophic lateral sclerosis (ALS): fast-fatigable (FF) MNs are more vulnerable than fast fatigue-resistant (FR) or slow (S) MNs. The reason for this selective vulnerability remains enigmatic. We report here that the extracellular matrix (ECM) protein osteopontin (OPN) is selectively expressed by FR and S MNs and ALS-resistant motor pools, whereas matrix metalloproteinase-9 (MMP-9) is selectively expressed by FF MNs. OPN is secreted and accumulated as extracellular granules in ECM in three ALS mouse models and a human ALS patient. In SOD1G93A mice, OPN/MMP-9 double positivity marks remodeled FR and S MNs destined to compensate for lost FF MNs before ultimately dying. Genetic ablation of OPN in SOD1G93A mice delayed disease onset but then accelerated disease progression. OPN induced MMP-9 up-regulation via αvβ3 integrin in ChAT-expressing Neuro2a cells, and also induced CD44-mediated astrocyte migration and microglial phagocytosis in a non-cell-autonomous manner. Our results demonstrate that OPN expressed by FR/S MNs is involved in the second-wave neurodegeneration by up-regulating MMP-9 through αvβ3 integrin in the mouse model of ALS. The differences in OPN/MMP-9 expression profiles in MN subsets partially explain the selective MN vulnerability in ALS.

L-type voltage-operated calcium channels contribute to astrocyte activation In vitro.

We have found a significant upregulation of L-type voltage-operated Ca(++) channels (VOCCs) in reactive astrocytes. To test if VOCCs are centrally involved in triggering astrocyte reactivity, we used in vitro models of astrocyte activation in combination with pharmacological inhibitors, siRNAs and the Cre/lox system to reduce the activity of L-type VOCCs in primary cortical astrocytes. The endotoxin lipopolysaccharide (LPS) as well as high extracellular K(+) , glutamate, and ATP promote astrogliosis in vitro. L-type VOCC inhibitors drastically reduce the number of reactive cells, astrocyte hypertrophy, and cell proliferation after these treatments. Astrocytes transfected with siRNAs for the Cav1.2 subunit that conducts L-type Ca(++) currents as well as Cav1.2 knockout astrocytes showed reduce Ca(++) influx by ∼80% after plasma membrane depolarization. Importantly, Cav1.2 knock-down/out prevents astrocyte activation and proliferation induced by LPS. Similar results were found using the scratch wound assay. After injuring the astrocyte monolayer, cells extend processes toward the cell-free scratch region and subsequently migrate and populate the scratch. We found a significant increase in the activity of L-type VOCCs in reactive astrocytes located in the growing line in comparison to quiescent astrocytes situated away from the scratch. Moreover, the migration of astrocytes from the scratching line as well as the number of proliferating astrocytes was reduced in Cav1.2 knock-down/out cultures. In summary, our results suggest that Cav1.2 L-type VOCCs play a fundamental role in the induction and/or proliferation of reactive astrocytes, and indicate that the inhibition of these Ca(++) channels may be an effective way to prevent astrocyte activation. GLIA 2016. GLIA 2016;64:1396-1415.

Integrin-mediated transactivation of P2X7R via hemichannel-dependent ATP release stimulates astrocyte migration

Our previous reports indicate that ligand-induced αVβ3 integrin and Syndecan-4 engagement increases focal adhesion formation and migration of astrocytes. Additionally, ligated integrins trigger ATP release through unknown mechanisms, activating P2X7 receptors (P2X7R), and the uptake of Ca2+ to promote cell adhesion. However, whether the activation of P2X7R and ATP release are required for astrocyte migration and whether αVβ3 integrin and Syndecan-4 receptors communicate with P2X7R via ATP remains unknown. Here, cells were stimulated with Thy-1, a reported αVβ3 integrin and Syndecan-4 ligand. Results obtained indicate that ATP was released by Thy-1 upon integrin engagement and required the participation of phosphatidylinositol-3-kinase (PI3K), phospholipase-C gamma (PLCγ) and inositol trisphosphate (IP3) receptors (IP3R). IP3R activation leads to increased intracellular Ca2+, hemichannel (Connexin-43 and Pannexin-1) opening, and ATP release. Moreover, silencing of the P2X7R or addition of hemichannel blockers precluded Thy-1-induced astrocyte migration. Finally, Thy-1 lacking the integrin-binding site did not stimulate ATP release, whereas Thy-1 mutated in the Syndecan-4-binding domain increased ATP release, albeit to a lesser extent and with delayed kinetics compared to wild-type Thy-1. Thus, hemichannels activated downstream of an αVβ3 integrin-PI3K-PLCγ-IP3R pathway are responsible for Thy-1-induced, hemichannel-mediated and Syndecan-4-modulated ATP release that transactivates P2X7Rs to induce Ca2+ entry. These findings uncover a hitherto unrecognized role for hemichannels in the regulation of astrocyte migration via P2X7R transactivation induced by integrin-mediated ATP release.

Activation of the KCa3.1 channel contributes to traumatic scratch injury-induced reactive astrogliosis through the JNK/c-Jun signaling pathway

Reactive astrogliosis is widely considered to contribute to pathogenic responses to stress and brain injury and to diseases as diverse as ischemia and neurodegeneration. We previously found that expression of the intermediate-conductance calcium-activated potassium channel (KCa3.1) involved in TGF-β-activated astrogliosis. In the present study, we investigated whether migration of cortical astrocytes following mechanical scratch injury involves the KCa3.1 channel, which contributes to Ca2+-mediated migration in other cells. We found that scratch injury increased the expression of KCa3.1 protein in reactive astrocytes. Application of the KCa3.1 blocker TRAM-34 decreased glial fibrillary acidic protein (GFAP) expression and slowed migration in a concentration-dependent manner. Application of the Ca2+ chelators, EGTA and BAPTA-AM, also slowed the migration of astrocytes. Blockade or genetic deletion of KCa3.1 both slowed and dramatically reduced the scratch injuries induced the sharp rise in astrocytes Ca2+ concentrations. The scratch injury-induced phosphorylation of JNK and c-Jun proteins was also attenuated both by blockade of KCa3.1 with TRAM-34 and in KCa3.1−/− astrocytes. Using KCa3.1 knockout mice, we further confirmed that deletion of KCa3.1 reduced expression of GFAP in an in vivo stab wound model. Taken together, our findings highlight a novel role for KCa3.1 in phenotypic modulation of reactive astrocytes and in astrocyte mobilization in response to mechanical stress, providing a potential target for therapeutic intervention in brain injuries.

The EBI2 signalling pathway plays a role in cellular crosstalk between astrocytes and macrophages.

EBI2 is a G protein-coupled receptor activated by oxysterol 7α, 25-dihydroxycholesterol (7α25HC) and regulates T cell-dependant antibody response and B cell migration. We recently found EBI2 is expressed in human astrocytes, regulates intracellular signalling and modulates astrocyte migration. Here, we report that LPS treatment of mouse astrocytes alters mRNA levels of EBI2 and oxysterols suggesting that the EBI2 signalling pathway is sensitive to LPS-mediated immune challenge. We also find that conditioned media obtained from LPS-stimulated mouse astrocytes induces macrophage migration, which is inhibited by the EBI2 antagonist NIBR189. These results demonstrate a role for the EBI2 signalling pathway in astrocytes as a sensor for immune challenge and for communication with innate immune cells such as macrophages.

Astrocyte migration in injectable gelatin-hydroxyphenyl propionic acid matrices for neuronal guidance in spinal cord injury

Event Abstract Back to Event Astrocyte migration in injectable gelatin-hydroxyphenyl propionic acid matrices for neuronal guidance in spinal cord injury Josna Joseph1*, Kazuhide Hayakawa2*, Motoichi Kurisawa3*, Li Shan Wang3*, Wanting Niu1 and Myron Spector1 1 VA Boston Healthcare System, Brigham&Women's Hospital, Harvard Medical School, Tissue Engineering, United States 2 Massachusetts General Hospital, Neuroprotection Research laboratory, Department of Neurology& Radiology, United States 3 Institute for Bioengineering and Nanotechnology, Singapore Introduction: The glial scar that is formed around residual cavities after certain spinal cord injuries is a physical and physiological impediment to neuronal regeneration. Gelatin–hydroxyphenyl propionic acid (Gtn-HPA), a novel injectable matrix with tuneable cross-linking properties[1], could provide the missing stroma in such defects, enabling migration of astrocytes from the glial scar into the matrix-filled defect and their guidance of neurons into the lesion[2]. The principal objective of this study was to evaluate astrocyte migration and activation in an in vitro ‘tissue simulant’ model using Gtn-HPA incorporating select growth factors. Materials and Methods: Cortical astrocytes were isolated from postnatal rats, and grown in DMEM high glucose medium. Astrocyte proliferation in Gtn-HPA gels at days 2, 4 and 7 was analysed by counting live cells stained by Calcein-AM under fluorescence microscopy. A three-dimensional migration assay model was set up in 3% agarose-coated 24-well culture plates by surrounding Gtn-HPA cores (1cm diameter) containing EGF (50 or 200 ng/ul) or TGF-β1 (50 or 200 ng/ul) with an annular tissue simulant of 2 wt. % Gtn-HPA gel (cross-linked with 0.1 U/ml horseradish peroxidase and 1.2 mM H202) containing 3*105 cells/ml. Cellular migration into the growth factor-laden inner core was analysed quantitatively by counting cells and measuring the migration distance (n=5 fields) from the gel interface to the farthest cell body (n=4 gels) under phase contrast microscopy. Immunostaining of 30-mm thick cryo sections of gel construct was carried out to detect the presence of GFAP-positive astrocytes. GFAP expression by migrated cells was quantified by Western blot by lysing the gel with collagenase 1 (500U/ml). The results were tested for statistical significance by ANOVA and post-hoc tests. Results and Discussion: Astrocytes migrated in response to chemotactic gradients in Gtn-HPA gels in contrast to the blank gels which displayed virtually no migration. The migration distance was significantly higher (p<0.001) in TGF-50 ng (TGFL) group when compared to EGF and blank Gtn-HPA control group (Fig.1). The number of cells infiltrating the inner core and GFAP expression was significantly higher in the EGF-50 ng (EGFL) group (Fig.2). This indicates that astrocytes migrate well in response to TGF-β1 and are activated by EGF at low concentration, whereas higher conc. of EGF has no specific effect on GFAP expression. Conclusion: This study shows that growth factor-laden Gtn-HPA hydrogels provide a supportive environment for astrocyte migration, proliferation and activation. Gtn-HPA hydrogels laden with optimum combination of EGF and TFG-β1 could effectively guide astrocyte infiltration and subsequent neurite outgrowth in a spinal cord injury defect with diminished glial scarring. J. Joseph acknowledges Fulbright Fellowship from United States India Educational Foundation. Gtn-HPA was provided by the Institute of Bioengineering and Nanotechnology, Singapore.

Depletion of kinesin-12, a myosin-IIB-interacting protein, promotes migration of cortical astrocytes.

Kinesin-12 (also named Kif15) participates in important events during neuronal development, such as cell division of neuronal precursors, migration of young neurons and establishment of axons and dendritic arbors, by regulating microtubule organization. Little is known about the molecular mechanisms behind the functions of kinesin-12, and even less is known about its roles in other cell types of the nervous system. Here, we show that kinesin-12 depletion from cultured rat cortical astrocytes decreases cell proliferation but increases migration. Co-immunoprecipitation, GST pulldown and small interfering RNA (siRNA) experiments indicated that kinesin-12 directly interacts with myosin-IIB through their tail domains. Immunofluorescence analyses indicated that kinesin-12 and myosin-IIB colocalize in the lamellar region of astrocytes, and fluorescence resonance energy transfer analyses revealed an interaction between the two. The phosphorylation at Thr1142 of kinesin-12 was vital for their interaction. Loss of their interaction through expression of a phosphorylation mutant of kinesin-12 promoted astrocyte migration. We suggest that kinesin-12 and myosin-IIB can form a hetero-oligomer that generates force to integrate microtubules and actin filaments in certain regions of cells, and in the case of astrocytes, that this interaction can modulate their migration.