Induces Astrocyte Proliferation Research Articles

Abstract TP30: Exercise Postconditioning After Ischemic Stroke: Neuroprotection Through NGF/TRPV1-Mediated Astrocyte-Neuron Interaction

Introduction: Astrocytes activation and interaction with neurons potentially affect stroke outcomes. Transient receptor potential vanilloid 1 (TRPV1) participated in reducing brain injury and nerve growth factor (NGF) affected TRPV1 activation. The present study investigated neuroprotection induced by exercise postconditioning (PostE) and elucidated the mechanistic regulation of astrocytes-neuron crosstalk via NGF/TRPV1 pathway. Methods: Adult Sprague-Dawley rats received 2 h middle cerebral artery (MCA) occlusion, followed by 24 h of reperfusion. The treadmill exercise was then initiated 24 h after reperfusion for 3 days (PostE). Brain damage was determined by infarct volume, edema and apoptotic proteins expression (Bcl-2, BAX and Caspase-3). Neuroplasticity was determined by dendritic spine staining and protein expression of BDNF, GAP-43, PSD-95 and synaptophysin. Neuronal TRPV1 expression, astrocyte proliferation and astrocytic NGF expression was assessed with immunofluorescence. Primary astrocyte-neuron Transwell cultivation were used to determine the secretion of astrocytic NGF activating neural TRPV1. Interaction between TRPV1 and NGF or TRPV1 and A-kinase anchoring protein150 (AKAP150) were detected by immunofluorescence and Co-IP. Results: PostE significantly decreased brain damage and induced neuroplasticity in ischemic rats. PostE induced astrocyte proliferation and astrocytic NGF secretion, which, in turn, activated TRPV1 expression in neuron. Either NGF or TRPV1 inhibitor eliminated the neuroplasticity, while NGF inhibitor decreased TRPV1 expression, suggesting the potential regulating role of astrocytic NGF on TRPV1. Primary cells cultivation demonstrated a NGF/TRPV1 and TRPV1/AKAP150 interplays showing astrocyte-neuron communication. Conclusion: PostE induced neuroprotection, which was mediated by the activation of astrocytes and their interaction with neurons through the NGF/TRPV1 pathway, highlighting the significance of astrocyte-neuron crosstalk in post-stroke recovery processes.

Glucose and Several Mitogenic Agents Modulate the Glucagon-Like Peptide-2 Receptor Expression in Cultured Rat Astrocytes.

Glucagon-like peptide-2 (GLP-2) is an intestinal trophic factor that induces astrocyte proliferation through its own receptor (GLP-2R), but the control of its expression is not well known. To study the effects of glucose and of different mitogenic agents on the control of GLP-2R expression in cultured rat astrocytes. GLP-2R mRNA content was measured by quantitative RT-PCR. GLP-2R expression was higher in proliferating than in resting cells. The expression was dependent of glucose concentration both in the absence and in the presence of GLP-2. In the presence of a high glucose concentration, GLP-2, PDGF, and PDGF plus GLP-2 presented opposite effects depending on the incubation time. However, insulin, IGF-1, and EGF alone, and plus GLP-2 had no effect. IGF-2, but not IGF-2 plus GLP-2, increased the expression. On the contrary, NGF decreased the GLP-2R expression, but NGF plus GLP-2 increased it even until values similar to those obtained with GLP-2 alone. Interestingly, in the presence of a low glucose concentration, leptin and NPY produced a significant reduction of GLP-2R expression. Astrocytes are distributed throughout the brain, where GLP-2 appears to have important functions. Since these cells express the GLP-2R, the results of this study could be considered of interest to advance the knowledge of the role of GLP-2 signaling in the CNS, which should lead a better understanding of the events that occur under normal and pathophysiological conditions.

Exposure to short-chain chlorinated paraffins induces astrocyte activation via JAK2/STAT3 signaling pathway

In the last few decades, short-chain chlorinated paraffins (SCCPs) have become the most heavily produced monomeric organohalogen compounds, and have been reported to induce multiple organ toxicity. However, the effects of SCCPs on the central nervous system are unknown. In the present study, we show that SCCP exposure induced astrocyte proliferation and increased the expression of two critical markers of astrocyte activation, glial fibrillary acidic protein and inducible nitric oxide synthase, in vivo and in vitro. SCCP exposure also increased inflammatory factory gene expression. Moreover, SCCP treatment triggered Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signalling, as shown by increased phosphorylation and STAT3 translocation to the nucleus. Both JAK2 and STAT3 inhibition effectively attenuated SCCP-induced astrocyte activation. Finally, JAK2 inhibition significantly rescued STAT3 phosphorylation and nuclear translocation. Taken together, JAK2/STAT3 pathway activation contributed to SCCP-induced astrocyte activation. These data will help elucidate the molecular mechanism underlying SCCP-induced neurotoxicity.

Mirtazapine exerts astrocyte-mediated dopaminergic neuroprotection

Mirtazapine, a noradrenergic and specific serotonergic antidepressant (NaSSA), is known to activate serotonin (5-HT) 1A receptor. Our recent study demonstrated that stimulation of astrocytic 5-HT1A receptors promoted astrocyte proliferation and upregulated antioxidative property in astrocytes to protect dopaminergic neurons against oxidative stress. Here, we evaluated the neuroprotective effects of mirtazapine against dopaminergic neurodegeneration in models of Parkinson’s disease (PD). Mirtazapine administration attenuated the loss of dopaminergic neurons in the substantia nigra and increased the expression of the antioxidative molecule metallothionein (MT) in the striatal astrocytes of 6-hydroxydopamine (6-OHDA)-injected parkinsonian mice via 5-HT1A receptors. Mirtazapine protected dopaminergic neurons against 6-OHDA-induced neurotoxicity in mesencephalic neuron and striatal astrocyte cocultures, but not in enriched neuronal cultures. Mirtazapine-treated neuron-conditioned medium (Mir-NCM) induced astrocyte proliferation and upregulated MT expression via 5-HT1A receptors on astrocytes. Furthermore, treatment with medium from Mir-NCM-treated astrocytes protected dopaminergic neurons against 6-OHDA neurotoxicity, and these effects were attenuated by treatment with a MT-1/2-specific antibody or 5-HT1A antagonist. Our study suggests that mirtazapine could be an effective disease-modifying drug for PD and highlights that astrocytic 5-HT1A receptors may be a novel target for the treatment of PD.

Activation of G protein-coupled receptor 30 protects neurons by regulating autophagy in astrocytes.

Ischemic stroke leads to neuronal damage induced by excitotoxicity, inflammation, and oxidative stress. Astrocytes play diverse roles in stroke and ischemia-induced inflammation, and autophagy is critical for maintaining astrocytic functions. Our previous studies showed that the activation of G protein-coupled receptor 30 (GPR30), an estrogen membrane receptor, protected neurons from excitotoxicity. However, the role of astrocytic GPR30 in maintaining autophagy and neuroprotection remained unclear. In this study, we found that the neuroprotection induced by G1 (GPR30 agonist) in wild-type mice after a middle cerebral artery occlusion was completely blocked in GPR30 conventional knockout (KO) mice but partially attenuated in astrocytic or neuronal GPR30 KO mice. In cultured primary astrocytes, glutamate exposure induced astrocyte proliferation and decreased astrocyte autophagy by activating mammalian target of rapamycin (mTOR) and c-Jun N-terminal kinase (JNK) and inhibiting p38 mitogen-activated protein kinase (MAPK) pathway. G1 treatment restored autophagy to its basal level by regulating the p38 pathway but not the mTOR and JNK signaling pathways. Our findings revealed a key role of GPR30 in neuroprotection via the regulation of astrocyte autophagy and support astrocytic GPR30 as a potential drug target against ischemic brain damage.

CB2R induces a protective response for epileptic seizure via the PI3K 110α-AKT signaling pathway.

Epilepsy is a chronic brain disease caused by abnormal discharging in the brain, which induces momentary brain dysfunction. Cannabinoid 2 receptor (CB2R) is expressed in central nervous system (CNS) and serves an important role in the pathogenesis of CNS diseases. The aim of the present study was to explore the effects of CB2R activation on phosphoinositide 3-kinase (PI3K) 110α-protein kinase B (AKT) signaling in an astrocyte model of epilepsy. Rat CTX TNA2 astrocytes were treated with Mg free solution to establish a cell model of epilepsy and were subsequently treated with a CB2R agonist (JWH133) and antagonist (AM630). Cell cycle analysis revealed that treatment using Mg free solution inhibited cell cycle transition. JWH133 facilitated cell cycle progression while AM630 inhibited it. Western blotting results demonstrated that treatment with Mg free solution downregulated the expression of cyclin D1, cyclin E, phosphorylated Retinoblastoma (p-Rb), B-cell lymphoma 2 (Bcl-2), PI3K 110α, p-AKT and p-mammalian target of rapamycin, whereas JWH133 treatment upregulated these proteins. AM630 ameliorated the JWH133-induced upregulation of these proteins. To confirm the involvement of AKT signaling, the AKT inhibitor wortmannin was used. The results revealed that wortmannin inhibited the effect of JWH133 on p-AKT, cyclin D1, p-Rb and Bcl-2 expression. In addition, the effects of JWH133 and AM630 on PI3K 110α-AKT signaling were verified using a rat model of epilepsy. In conclusion, the present study demonstrates that CB2R activation induces astrocyte proliferation and survival via activation of the PI3K 110α-AKT signaling pathway.

Novel celecoxib analogues inhibit glial production of prostaglandin E2, nitric oxide, and oxygen radicals reverting the neuroinflammatory responses induced by misfolded prion protein fragment 90-231 or lipopolysaccharide

We tested the efficacy of novel cyclooxygenase 2 (COX-2) inhibitors in counteracting glia-driven neuroinflammation induced by the amyloidogenic prion protein fragment PrP90-231 or lipopolysaccharide (LPS). In search for molecules with higher efficacy than celecoxib, we focused our study on its 2,3-diaryl-1,3-thiazolidin-4-one analogues. As experimental models, we used the immortalized microglial cell line N9, rat purified microglial primary cultures, and mixed cultures of astrocytes and microglia. Microglia activation in response to PrP90-231 or LPS was characterized by growth arrest, morphology changes and the production of reactive oxygen species (ROS). Moreover, PrP90-231 treatment caused the overexpression of the inducible nitric oxide synthase (iNOS) and COX-2, with the consequent nitric oxide (NO), and prostaglandin E2 (PGE2) accumulation. These effects were challenged by different celecoxib analogues, among which Q22 (3-[4-(sulfamoyl)phenyl]-2-(4-tolyl)thiazolidin-4-one) inhibited microglia activation more efficiently than celecoxib, lowering both iNOS and COX-2 activity and reducing ROS release. During neurodegenerative diseases, neuroinflammation induced by amyloidogenic peptides causes the activation of both astrocytes and microglia with these cell populations mutually regulating each other. Thus the effects of PrP90-231 and LPS were also studied on mixed glial cultures containing astrocytes and microglia. PrP90-231 treatment elicited different responses in the co-cultures induced astrocyte proliferation and microglia growth arrest, resulting in a differential ability to release proinflammatory molecules with the production of NO and ROS mainly attributable on microglia, while COX-2 expression was induced also in astrocytes. Q22 effects on both NO and PGE2 secretion were more significant in the mixed glial cultures than in purified microglia, demonstrating Q22 ability to revert the functional interaction between astrocytes and microglia. These results demonstrate that Q22 is a powerful drug able to revert glial neuroinflammatory responses and might represent a lead to explore the chemical space around celecoxib frameworks to design even more effective agents, paving the way to novel approaches to contrast the neuroinflammation-dependent toxicity.

The Expression of CUGBP1 After Spinal Cord Injury in Rats.

CUG-binding protein 1, a member of the CELF (CUGBP and embryonic lethal abnormal vision-like factor) family of RNA-binding proteins, is shown to be multifunctional, regulating many posttranscriptional processes including alternative splicing, deadenylation, mRNA decay, and translation. Recently, CUGBP1 is found to represses p27 IRES activity and inhibits expression of endogenous p27 in cultured breast cancer cells. However, the roles of CUGBP1 in central nervous system injury remain unknown. In our study, we performed acute spinal cord injury (SCI) model in adult rats in order to research the expression changes of CUGBP1 in spinal cord. Western blot analysis showed a marked upregulation of CUGBP1 after SCI. Immunohistochemistry analysis revealed a wide distribution of CUGBP1 in the spinal cord. Double immunofluorescence staining indicated that CUGBP1 immunoreactivity was increased predominantly in neurons and astrocytes after SCI. Moreover, colocalization of CUGBP1/proliferating cell nuclear antigen (PCNA) was detected in GFAP positive cells. We also examined the expression profiles of p27, which was up-regulated after SCI. To further understand whether CUGBP1 plays a role in astrocyte proliferation, we applied LPS to induce astrocyte proliferation in vitro. Western blot analysis demonstrated that CUGBP1 expression was positively correlated with PCNA expression, and the p27 expression was negatively correlated with CUGBP1 expression following LPS stimulation. Our results suggest that CUGBP1 might be implicated in the pathophysiology of spinal cord after SCI.

Crosstalk between macrophages and astrocytes affects proliferation, reactive phenotype and inflammatory response, suggesting a role during reactive gliosis following spinal cord injury.

BackgroundLarge-scale macrophage infiltration and reactive astrogliosis are hallmarks of early spinal cord injury (SCI) pathology. The exact nature of the macrophage response and relationship between these phenomena have not been explored in detail. Here, we have investigated these responses using a combination of in vivo SCI models, organotypic and primary cultures.MethodsIn vivo macrophage response was investigated using a contusive injury mouse model. Interactions between astrocytes and macrophages were studied in primary or organotypic cultures. Proliferation was assessed though MTT assay and nucleotide incorporation and gene expression changes through qPCR.ResultsSeven days following contusive SCI, a mixed M1/M2 macrophage response was seen in the injury site. Conditioned medium from primary M1, but not M2, macrophages are able to induce astrocyte proliferation in both organotypic spinal cord cultures and primary astrocytes. Soluble factors from M1 macrophages induce a reactive astrocyte gene expression pattern, whereas M2 factors inhibit expression of these genes. M2-stimulated astrocytes are also able to decrease both M1 and M2 macrophage proliferation and decrease TNFα production in M1 macrophages.ConclusionsThese results suggest a strong role of M1 macrophages in inducing reactive astrogliosis and the existence of an astrocyte-mediated negative feedback system in order to dampen the immune response. These results, combined with the poor outcomes of the current immunosuppressive steroid treatments in SCI, indicate the need for more targeted therapies, taking into account the significantly different effects of M1 and M2 macrophages, in order to optimise outcome.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-015-0327-3) contains supplementary material, which is available to authorized users.

Spatiotemporal Profile and Essential Role of RBM3 Expression after Spinal Cord Injury in Adult Rats

Hypoxia and other adverse conditions are usually encountered by rapidly growing cells. The RNA-binding motif protein 3 (RBM3) is induced by low temperature and hypoxia. However, its expression and function in spinal cord injury are still unclear. To investigate the certain expression and biological function in the central nervous system, we performed an acute spinal cord contusion injury (SCI) model in adult rats. Western blot analysis indicated a striking expression upregulation of RBM3 after spinal cord injury (SCI). Double immunofluorescence staining prompted that RBM3 immunoreactivity was found in astrocytes and neurons. Interestingly, RBM3 expression was increased predominantly in astrocytes. Furthermore, colocalization of RBM3 with proliferating cell nuclear antigen (PCNA) was detected in astrocytes. To further understand whether RBM3 plays a role in astrocyte proliferation, we applied lipopolysaccharide (LPS) to induce astrocyte proliferation in vitro. Western blot analysis demonstrated that RBM3 expression was positively correlated with PCNA expression following LPS stimulation. Immunofluorescence analysis showed that the expression of RBM3 was also changed following the stimulation of astrocytes with LPS, which was parallel with the data in vivo. Additionally, knocking RBM3 down with small interfering RNA (siRNA) demonstrated that RBM3 might play a significant role in the proliferation of astrocytes treated by hypoxia in vitro. These results suggest that RBM3 may be involved in the proliferation of astrocytes after SCI. To summarize, we firstly uncover the temporal and spatial expression changes of RBM3 in spinal cord injury. Our data suggest that RBM3 might be implicated in central nervous system pathophysiology after SCI.

Leukemia inhibitory factor inhibits the proliferation of primary rat astrocytes induced by oxygen-glucose deprivation.

Leukemia inhibitory factor (LIF) is a neuroprotective cytokine that is necessary for the normal development of astrocytes. Oxygen-glucose deprivation (OGD) can induce astrocyte proliferation by increasing hypoxia-inducible factor alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF). Here, we studied whether LIF affects the proliferation of cultured primary rat astrocytes under OGD conditions by measuring EdU incorporation into astrocyte DNA and the expression of proliferating cell nuclear antigen (PCNA) mRNA and protein. Our findings show that low concentrations of LIF (5 and 10 ng/mL) significantly decreased EdU incorporation and downregulated the expression of PCNA mRNA and PCNA protein in astrocytes subjected to OGD. A low concentration of LIF (10 ng/mL) clearly inhibited astrocyte proliferation induced by OGD, while a higher concentration (50 ng/mL) had no effect. To investigate the mechanism of this inhibition by LIF (10 ng/mL), the expression of 3 related genes (LIF receptor, HIF-1alpha, and VEGF) was assessed using real-time PCR; VEGF protein expression was measured by Western blot. Our results indicate that LIFR mRNA was downregulated in astrocytes subjected to OGD. Interestingly, treatment with LIF further reduced LIFR mRNA expression in these cells. LIF treatment also decreased the expression of HIF-1alpha mRNA, VEGF mRNA, and VEGF protein induced by OGD. Low concentrations of LIF were observed to inhibit astrocyte proliferation induced by OGD.

Targeting 5-HT1A receptors in astrocytes to protect dopaminergic neurons in parkinsonian models

Astrocytes are abundant neuron-supporting glial cells that harbor a powerful arsenal of neuroprotective antioxidative molecules and neurotrophic factors. Here we examined whether enrichment with healthy striatal astrocytes can provide neuroprotection against progressive dopaminergic neurodegeneration. Serotonin 1A (5-HT1A) agonist 8-OH-DPAT induced astrocyte proliferation and increased metallothionein-1/-2 (MT-1/-2), antioxidative molecules, in cultured astrocytes and the striatum of mice. Primary cultured mesencephalic dopamine neurons were protected against oxidative stress by preincubation with conditioned media from 8-OH-DPAT-treated astrocytes. These protective effects were canceled by 5-HT1A antagonist or MT-1/-2-specific antibody. Furthermore, reduction of nigrostriatal dopaminergic neurons in 6-hydroxydopamine-lesioned parkinsonian model mice was significantly abrogated by repeated injections of 8-OH-DPAT. Treatment with 8-OH-DPAT markedly increased the expression of MT in striatal astrocytes in the hemi-parkinsonian mice. Our study provides a promising therapeutic strategy of neuroprotection against oxidative stress and progressive dopaminergic neurodegeneration by demonstrating the efficacy of targeting 5-HT1A receptors in astrocytes.

Fibroblast Growth Factor Inducible (Fn14)-specific Antibodies Concomitantly Display Signaling Pathway-specific Agonistic and Antagonistic Activity

Fn14 is a therapeutic target in various diseases. Anti-Fn14 antibodies activate the alternative NFκB pathway but not other Fn14-related activities induced by soluble or membrane-bound TWEAK. FcγR-bound anti-Fn14 antibodies, however, activate the full spectrum of Fn14-associated activities. Anti-Fn14 antibodies elicit agonistic activities differing from those of the natural Fn14 ligand TWEAK. These findings influence the rationale of designing Fn14-targeted therapies. The Fn14-specific monoclonal antibodies PDL192 and P4A8, which are under consideration in clinical trials, showed no agonistic activity with respect to IL8 production and cell death induction. However, oligomerization with protein G or binding to Fcγ receptors converted both anti-Fn14 antibodies into potent agonists. TNF-like weak inducer of apoptosis (TWEAK), the ligand of Fn14, occurs naturally in two forms with partly different signaling capabilities, as a membrane-bound ligand and as a soluble trimeric molecule. Although membrane TWEAK strongly triggers all Fn14-associated pathways, soluble TWEAK predominately triggers the alternative nuclear factor κB (NFκB) pathway and enhances TNF-induced cell death but has only a poor effect on the classical NFκB pathway and chemokine production. Thus, the oligomerized and FcγR-bound anti-Fn14 mAbs mimicked the activity of membrane TWEAK. Notably, both anti-Fn14 antibodies significantly triggered p100 processing, the hallmark of the alternative NFκB pathway, and therefore resembled soluble TWEAK. In contrast to the latter, however, the anti-Fn14s showed no effect on TNF receptor 1-induced cell death and P4A8 even blocked the corresponding TWEAK response. Thus, we showed that Fn14 antibodies display an alternative NFκB pathway-specific agonistic activity but fail to phenocopy other activities of soluble TWEAK, whereas oligomerized or FcγR-bound Fn14 antibodies fully mimic the activity of membrane TWEAK. In view of the trivalent nature of the TWEAK-Fn14 interaction, this suggests that the alternative NFκB pathway is uniquely responsive already to Fn14 dimerization enabling antibodies to elicit an unnatural response pattern distinct from that of the naturally occurring Fn14 ligands.

Angiotensin III Induces c-Jun N-terminal Kinase Leading to Proliferation of Rat Astrocytes

Previously, we showed in cultured rat astrocytes that angiotensin (Ang) III induced astrocyte proliferation and phosphorylation of ERK1/2 mitogen activated protein (MAP) kinases through interaction with the AT(1) receptor. In the current study, we determined whether the c-Jun N terminal kinase (JNK) MAP kinase pathway was similarly affected by the peptide in cultured brainstem astrocytes. Ang III induced JNK phosphorylation in a concentration- and time-dependent manner. Similar to ERK1/2 phosphorylation, maximal phosphorylation occurred with 100 nM Ang III and was apparent within a minute of exposure to the peptide. Peak effects were observed over a 5-15 min time range. Pretreatment of brainstem astrocytes with the JNK inhibitor, SP600125, prevented Ang III phosphorylation of JNK, as well as Ang III-mediated astrocyte growth. The selective AT(1) receptor antagonist, Losartan, prevented Ang III-induced JNK phosphorylation. Pretreatment of astrocytes with the AT(2) receptor blocker PD123319 was ineffective in preventing JNK phosphorylation by Ang III. Interestingly, both Ang II and Ang III induced JNK phosphorylation to a similar extent suggesting that the two peptides were equipotent in this effect. Our findings suggest that Ang III interacts with Ang AT(1) receptors to directly stimulate the JNK MAP kinase pathway leading to astrocyte growth. This study is the first to show that Ang III actions may involve the JNK MAP kinase pathway in astrocytes and provide key information that may lead to a better understanding of the functions of Ang III in the central nervous system, in particular in astrocytes.

Angiotensin III stimulates ERK1/2 mitogen-activated protein kinases and astrocyte growth in cultured rat astrocytes

Angiotensin (Ang) III is a biologically active metabolite of Ang II with similar effects and receptor binding properties as Ang II. Most Ang III studies delineate physiological effects of the peptide but, the intracellular pathways leading to the actions are unknown and are a focus of these studies. We investigated in cultured brainstem and cerebellum rat astrocytes whether Ang III stimulates ERK1/2 mitogen activated protein (MAP) kinases and astrocyte growth. Ang III significantly stimulated ERK1/2 MAP kinases in a dose- and time-dependent manner. The maximal stimulation occurred with 100 nM Ang III (2.8±0.3 and 2.3±0.1-fold over basal, in brainstem and cerebellum astrocytes, respectively). This stimulation occurred as early as 1 min, and was sustained for at least 15 min. Moreover, inhibition of the ERK1/2 MAP kinase pathway by 10 μM PD98059 attenuated Ang III-induced ERK1/2 phosphorylation. Ang III induction of ERK1/2 occurred via stimulation of the Ang AT(1) receptor since pretreatment with 10 μM Losartan, a selective AT(1) receptor blocker, prevented Ang III-induced ERK1/2 phosphorylation. The selective AT(2) Ang receptor blocker PD123319 was ineffective. Comparable to Ang II, Ang III also stimulated astrocyte growth in a concentration-dependent manner, an effect that occurred via activation of the AT(1) receptor as well. These findings suggest that Ang III has similar effects as Ang II in astrocytes since it rapidly stimulates the phosphorylation of the ERK1/2 MAP kinases and induces astrocyte proliferation through activation of the AT(1) receptor. These studies are important in establishing signaling pathways for Ang III and provide validation of the central role of Ang III.

FOXO3a inhibits TNF‐α‐ and IL‐1β‐induced astrocyte proliferation:Implication for reactive astrogliosis

Reactive astrogliosis is one of the pathological hallmarks of neurodegenerative diseases. Inflammatory cytokines, such as TNF-α and IL-1β, have been shown to mediate the reactive astrogliosis in neurodegenerative diseases; however, the molecular mechanism remains unclear. In this study, we investigated the role of transcription factor FOXO3a on astrocyte proliferation, one primary aspect of severe reactive astrogliosis. Our results confirmed that TNF-α and IL-1β increased astrocyte proliferation, as determined by Ki67 and BrdU immunostaining. Furthermore, we found that cytokine-mediated astrocyte proliferation was accompanied by an increase of the phosphorylation and reduced nuclear expression of FOXO3a. Intracranial injection of TNF-α and IL-1β induced astrocyte proliferation and hypertrophy, which was associated with reduced nuclear expression of Foxo3a in astrocytes. To determine the function of FOXO3a in astrocyte proliferation, wild type FOXO3a was overexpressed with adenovirus, which subsequently upregulated p27Kip1 and Gadd45α, and significantly inhibited cytokine-induced astrocyte proliferation. In contrast, overexpression of dominant negative FOXO3a decreased p27Kip1, upregulated cyclin D1 and promoted astrocyte proliferation. Along the same line, astrocytes isolated from Foxo3a-null mice have higher proliferative potential. In response to intracranial injection of cytokines, Foxo3a-null mice manifested severe astrogliosis in vivo. In conclusion, FOXO3a is important in restraining astrocyte proliferation during proinflammatory cytokine stimulation and loss of function of FOXO3a may be responsible for the proliferation of astrocytes in the severe form of reactive astrogliosis. Understanding the key regulatory role of FOXO3a in reactive astrogliosis may provide a novel therapeutic target during neuroinflammation.

KPC1 Expression and Essential Role After Acute Spinal Cord Injury in Adult Rat

KPC1 (Kip1 ubiquitylation-promoting complex 1) is the catalytic subunit of the ubiquitin ligase KPC, which regulates the degradation of the cyclin-dependent kinase inhibitor p27(kip1) at the G1 phase of the cell cycle. To elucidate the expression and role of KPC1 in nervous system lesion and repair, we performed an acute spinal cord contusion injury (SCI) model in adult rats. Western blot analysis showed a significant up-regulation of KPC1 and a concomitant down-regulation of p27(kip1) following spinal injury. Immunohistochemistry and immunofluorescence revealed wide expression of KPC1 in the spinal cord, including expression in neurons and astrocytes. After injury, KPC1 expression was increased predominantly in astrocytes, which highly expressed PCNA, a marker for proliferating cells. Co-immunoprecipitation demonstrated increased interactions between p27(kip1) and KPC1 4 days after injury. To understand whether KPC1 plays a role in astrocyte proliferation, we applied LPS to induce astrocyte proliferation in vitro. Western blot analysis demonstrated that p27(kip1) expression was negatively correlated with KPC1 expression following LPS stimulation. Immunofluorescence analysis showed subcellular localizations of p27(kip1) and KPC1 were also changed following the stimulation of astrocytes with LPS. These results suggest that KPC1 is related to the down-regulation of p27(kip1); this event may be involved in the proliferation of astrocytes after SCI.

Prostaglandin E2 released from activated microglia enhances astrocyte proliferation in vitro

Microglial activation has been implicated in many astrogliosis-related pathological conditions including astroglioma; however, the detailed mechanism is not clear. In this study, we used primary enriched microglia and astrocyte cultures to determine the role of microglial prostaglandin E 2 (PGE 2) in the proliferation of astrocytes. The proliferation of astrocytes was measured by BrdU incorporation. The level of PGE 2 was measured by ELISA method. Pharmacological inhibition or genetic ablation of COX-2 in microglia were also applied in this study. We found that proliferation of astrocytes increased following lipopolysaccharide (LPS) treatment in the presence of microglia. Furthermore, increased proliferation of astrocytes was observed in the presence of conditioned media from LPS-treated microglia. The potential involvement of microglial PGE 2 in enhanced astrocyte proliferation was suggested by the findings that PGE 2 production and COX-2 expression in microglia were increased by LPS treatment. In addition, activated microglia-induced increases in astrocyte proliferation were blocked by the PGE 2 antagonist AH6809, COX-2 selective inhibitor DuP-697 or by genetic knockout of microglial COX-2. These findings were further supported by the finding that addition of PGE 2 to the media significantly induced astrocyte proliferation. These results indicate that microglial PGE 2 plays an important role in astrocyte proliferation, identifying PGE 2 as a key neuroinflammatory molecule that triggers the pathological response related to uncontrollable astrocyte proliferation. These findings are important in elucidating the role of activated microglia and PGE 2 in astrocyte proliferation and in suggesting a potential avenue in the use of anti-inflammatory agents for the therapy of astroglioma.

Fn14-TRAIL, a Chimeric Intercellular Signal Exchanger, Attenuates Experimental Autoimmune Encephalomyelitis

Hallmarks of the pathogenesis of autoimmune encephalomyelitis include perivascular infiltration of inflammatory cells into the central nervous system, multifocal demyelination in the brain and spinal cord, and focal neuronal degeneration. Optimal treatment of this complex disease will ultimately call for agents that target the spectrum of underlying pathogenic processes. In the present study, Fn14-TRAIL is introduced as a unique immunotherapeutic fusion protein that is designed to exchange and redirect intercellular signals within inflammatory cell networks, and, in so doing, to impact multiple pathogenic events and yield a net anti-inflammatory effect. In this soluble protein product, a Fn14 receptor component (capable of blocking the pro-inflammatory TWEAK ligand) is fused to a TRAIL ligand (capable of inhibiting activated, pathogenic T cells). Sustained Fn14-TRAIL expression was obtained in vivo using a transposon-based eukaryotic expression vector. Fn14-TRAIL expression effectively prevented chronic, nonremitting, paralytic disease in myelin oligodendrocyte glycoprotein-challenged C57BL/6 mice. Disease suppression in this model was reflected by decreases in the clinical score, disease incidence, nervous tissue inflammation, and Th1, Th2, and Th17 cytokine responses. Significantly, the therapeutic efficacy of Fn14-TRAIL could not be recapitulated simply by administering its component parts (Fn14 and TRAIL) as soluble agents, either alone or in combination. Its functional pleiotropism was manifest in its additional ability to attenuate the enhanced permeability of the blood-brain barrier that typically accompanies autoimmune encephalomyelitis.

Endothelin-1 Regulates Astrocyte Proliferation and Reactive Gliosis via a JNK/c-Jun Signaling Pathway

Reactive gliosis is characterized by enhanced glial fibrillary acidic protein (GFAP) expression, cellular hypertrophy, and astrocyte proliferation. The cellular and molecular mechanisms underlying this process are still largely undefined. We investigated the role of endothelin-1 (ET-1) in reactive gliosis in corpus callosum after lysolecithin (LPC)-induced focal demyelination and in cultured astrocytes. We show that ET-1 levels are upregulated in demyelinated lesions within 5 d after LPC injection, together with enhanced astrocyte proliferation, GFAP expression, and JNK phosphorylation. Infusion of the pan-ET-receptor (ET-R) antagonist Bosentan or the selective ET(B)-R antagonist BQ788 into the corpus callosum prevented postlesion astrocyte proliferation and JNK phosphorylation. In cultured astrocytes, ET-1-induced activation of ET(B)-Rs promotes a reactive phenotype by enhancing both GFAP expression and astrocyte proliferation. In the same cells, ET-1 activates both JNK and p38MAPK pathways, and induces c-Jun expression at the mRNA and protein levels. By using selective pharmacological inhibitors, we also provide evidence that ET-1 induces astrocyte proliferation and GFAP expression through activation of ERK- and JNK-dependent pathways, consistent with the previous observation of ET-1-induced activation of ERK (Schinelli et al., 2001). Finally, we show by gain and loss of function that increased c-Jun expression enhances the proliferative response of astrocytes to ET-1, whereas c-jun siRNA prevents ET-1-induced cell proliferation. Our results indicate that the effects of ET-1 on astrocyte proliferation depend on c-Jun induction and activation through ERK- and JNK-dependent pathways, and suggest that ET-R-associated pathways might represent important targets to control reactive gliosis.