Shinrye Lee

Lipocalin-2 is an autocrine mediator of reactive astrocytosis.

Astrocytes, the most abundant glial cell type in the brain, provide metabolic and trophic support to neurons and modulate synaptic activity. In response to a brain injury, astrocytes proliferate and become hypertrophic with an increased expression of intermediate filament proteins. This process is collectively referred to as reactive astrocytosis. Lipocalin 2 (lcn2) is a member of the lipocalin family that binds to small hydrophobic molecules. We propose that lcn2 is an autocrine mediator of reactive astrocytosis based on the multiple roles of lcn2 in the regulation of cell death, morphology, and migration of astrocytes. lcn2 expression and secretion increased after inflammatory stimulation in cultured astrocytes. Forced expression of lcn2 or treatment with LCN2 protein increased the sensitivity of astrocytes to cytotoxic stimuli. Iron and BIM (Bcl-2-interacting mediator of cell death) proteins were involved in the cytotoxic sensitization process. LCN2 protein induced upregulation of glial fibrillary acidic protein (GFAP), cell migration, and morphological changes similar to characteristic phenotypic changes termed reactive astrocytosis. The lcn2-induced phenotypic changes of astrocytes occurred through a Rho–ROCK (Rho kinase)–GFAP pathway, which was positively regulated by nitric oxide and cGMP. In zebrafishes, forced expression of rat lcn2 gene increased the number and thickness of cellular processes in GFAP-expressing radial glia cells, suggesting that lcn2 expression in glia cells plays an important role in vivo. Our results suggest that lcn2 acts in an autocrine manner to induce cell death sensitization and morphological changes in astrocytes under inflammatory conditions and that these phenotypic changes may be the basis of reactive astrocytosis in vivo.

A Dual Role of Lipocalin 2 in the Apoptosis and Deramification of Activated Microglia

Activated microglia are thought to undergo apoptosis as a self-regulatory mechanism. To better understand molecular mechanisms of the microglial apoptosis, apoptosis-resistant variants of microglial cells were selected and characterized. The expression of lipocalin 2 (lcn2) was significantly down-regulated in the microglial cells that were resistant to NO-induced apoptosis. lcn2 expression was increased by inflammatory stimuli in microglia. The stable expression of lcn2 as well as the addition of rLCN2 protein augmented the sensitivity of microglia to the NO-induced apoptosis, while knockdown of lcn2 expression using short hairpin RNA attenuated the cell death. Microglial cells with increased lcn2 expression were more sensitive to other cytotoxic agents as well. Thus, inflammatory activation of microglia may lead to up-regulation of lcn2 expression, which sensitizes microglia to the self-regulatory apoptosis. Additionally, the stable expression of lcn2 in BV-2 microglia cells induced a morphological change of the cells into the round shape with a loss of processes. Treatment of primary microglia cultures with the rLCN2 protein also induced the deramification of microglia. The deramification of microglia was closely related with the apoptosis-prone phenotype, because other deramification-inducing agents such as cAMP-elevating agent forskolin, ATP, and calcium ionophore also rendered microglia more sensitive to cell death. Taken together, our results suggest that activated microglia may secrete LCN2 protein, which act in an autocrine manner to sensitize microglia to the self-regulatory apoptosis and to endow microglia with an amoeboid form, a canonical morphology of activated microglia in vivo.

Secreted protein lipocalin-2 promotes microglial M1 polarization

Activated macrophages are classified into two different forms: classically activated (M1) or alternatively activated (M2) macrophages. The presence of M1/M2 phenotypic polarization has also been suggested for microglia. Here, we report that the secreted protein lipocalin 2 (LCN2) amplifies M1 polarization of activated microglia. LCN2 protein (EC50 1 μg/ml), but not glutathione S‐transferase used as a control, increased the M1‐related gene expression in cultured mouse microglial cells after 8–24 h. LCN2 was secreted from M1‐polarized, but not M2‐polarized, microglia. LCN2 inhibited phosphorylation of STAT6 in IL‐4‐stimulated microglia, suggesting LCN2 suppression of the canonical M2 signaling. In the lipopolysaccharide (LPS)‐induced mouse neuroinflammation model, the expression of LCN2 was notably increased in microglia. Primary microglial cultures derived from LCN2‐deficient mice showed a suppressed M1 response and enhanced M2 response. Mice lacking LCN2 showed a markedly reduced M1‐related gene expression in microglia after LPS injection, which was consistent with the results of histological analysis. Neuroinflammation‐associated impairment in motor behavior and cognitive function was also attenuated in the LCN2‐deficient mice, as determined by the rotarod performance test, fatigue test, open‐field test, and object recognition task. These findings suggest that LCN2 is an M1‐amplifier in brain microglial cells.—Jang, E., Lee, S., Kim, J.‐H., Kim, J.‐H., Seo, J.‐W., Lee, W.‐H., Mori, K., Nakao, K., Suk, K. Secreted protein lipocalin‐2 promotes microglial M1 polarization. FASEB J. 27, 1176–1190 (2013). www.fasebj.org

Lipocalin-2 Is a chemokine inducer in the central nervous system: role of chemokine ligand 10 (CXCL10) in lipocalin-2-induced cell migration.

Background: LCN2 has been implicated in cell morphology and migration. Results: LCN2 promotes cell migration through up-regulation of chemokines (CXCL10) in brain both in vitro and in vivo. Conclusion: LCN2 is a chemokine inducer in the CNS and may accelerate cell migration under inflammatory conditions in an autocrine or paracrine manner. Significance: LCN2 could be targeted to therapeutically modulate glial responses in various neuroinflammatory disease conditions. The secreted protein lipocalin-2 (LCN2) has been implicated in diverse cellular processes, including cell morphology and migration. Little is known, however, about the role of LCN2 in the CNS. Here, we show that LCN2 promotes cell migration through up-regulation of chemokines in brain. Studies using cultured glial cells, microvascular endothelial cells, and neuronal cells suggest that LCN2 may act as a chemokine inducer on the multiple cell types in the CNS. In particular, up-regulation of CXCL10 by JAK2/STAT3 and IKK/NF-κB pathways in astrocytes played a pivotal role in LCN2-induced cell migration. The cell migration-promoting activity of LCN2 in the CNS was verified in vivo using mouse models. The expression of LCN2 was notably increased in brain following LPS injection or focal injury. Mice lacking LCN2 showed the impaired migration of astrocytes to injury sites with a reduced CXCL10 expression in the neuroinflammation or injury models. Thus, the LCN2 proteins, secreted under inflammatory conditions, may amplify neuroinflammation by inducing CNS cells to secrete chemokines such as CXCL10, which recruit additional inflammatory cells.

Phenotypic Polarization of Activated Astrocytes: the Critical Role of Lipocalin-2 in the Classical Inflammatory Activation of Astrocytes

Astrocytes provide structural and functional support for neurons, as well as display neurotoxic or neuroprotective phenotypes depending upon the presence of an immune or inflammatory microenvironment. This study was undertaken to characterize multiple phenotypes of activated astrocytes and to investigate the regulatory mechanisms involved. We report that activated astrocytes in culture exhibit two functional phenotypes with respect to pro- or anti-inflammatory gene expression, glial fibrillary acidic protein expression, and neurotoxic or neuroprotective activities. The two distinct functional phenotypes of astrocytes were also demonstrated in a mouse neuroinflammation model, which showed pro- or anti-inflammatory gene expression in astrocytes following challenge with classical or alternative activation stimuli; similar results were obtained in the absence of microglia. Subsequent studies involving recombinant lipocalin-2 (LCN2) protein treatment or Lcn2-deficient mice indicated that the pro- or anti-inflammatory functionally polarized phenotypes of astrocytes and their intracellular signaling pathway were critically regulated by LCN2 under in vitro and in vivo conditions. Astrocyte-derived LCN2 promoted classical proinflammatory activation of astrocytes but inhibited IL-4–STAT6 signaling, a canonical pathway involved in alternative anti-inflammatory activation. Our results suggest that the secreted protein LCN2 is an autocrine modulator of the functional polarization of astrocytes in the presence of immune or inflammatory stimuli and that LCN2 could be targeted therapeutically to dampen proinflammatory astrocytic activation and related pathologies in the CNS.

Regulation by lipocalin‐2 of neuronal cell death, migration, and morphology

A secreted protein, lipocalin‐2 (LCN2), has been previously shown to regulate a variety of cellular phenotypes such as cell death, migration, and morphology. The role of LCN2, however, appears to be different depending on the cellular context. Here, we investigated how LCN2 influences neuronal phenotypes by using primary cortical neuronal cell cultures and neuroblastoma cell lines as a model. When exposed to LCN2 protein, neurons and neuroblastoma cells were sensitized to cell death evoked by nitric oxide, oxidative stress, and tumor necrosis factor‐α (TNF‐α). A forced expression of lcn2 in glia enhanced neuronal cell death in cocultures of glia and neurons, indicating that both exogenous protein addition and endogenous expression of lcn2 give rise to similar results. Iron and BCL2‐interacting mediator of cell death (BIM) protein were involved in LCN2‐induced cell death sensitization, based on the studies using iron donor, chelator, siderophore, and short hairpin RNA (shRNA)‐mediated knockdown of bim expression. Furthermore, cell migration assay and immunofluorescence microscopic observation revealed that LCN2 accelerated neuronal motility and process extension, suggesting multiple roles for LCN2 in the regulation of neuronal cell death, migration, and morphology.

Gangliosides induce autophagic cell death in astrocytes

Background and purpose:  Gangliosides, sialic acid‐containing glycosphingolipids, abundant in brain, are involved in neuronal function and disease, but the precise molecular mechanisms underlying their physiological or pathological activities are poorly understood. In this study, the pathological role of gangliosides in the extracellular milieu with respect to glial cell death and lipid raft/membrane disruption was investigated.

Analysis of glial secretome: The long pentraxin PTX3 modulates phagocytic activity of microglia

Microglia, as the phagocytes of the central nervous system, play an important role in the recognition, engulfment, and clearance of apoptotic cells and invading microbes. Proteins secreted from activated glial cells may affect microglial phagocytic activity. Secreted proteins of mixed glial cells stimulated with lipopolysaccharide (LPS) and interferon-γ (IFN-γ) for 24h were identified for the first time by liquid chromatography and tandem mass spectrometric analysis. Several proteins were newly identified as a glia-secreted protein. Among the proteins identified by the mixed glia secretome analysis, pentraxin 3 (PTX3) secretion was most highly induced by LPS/IFN-γ stimulation. Expression of PTX3 mRNA was detected in primary microglia and astrocyte cultures as well as glial cell lines. Glial secretion of PTX3 and its inflammatory induction was confirmed by Western blot analysis of conditioned media of mixed glial cultures. PTX3 did not influence LPS-induced nitric oxide production or neurotoxicity of BV-2 microglial cells. Most importantly, PTX3 selectively modulated microglial phagocytosis activity; it promoted engulfment of zymosan particles, while it inhibited uptake of apoptotic cells. Our results indicate that glia-derived PTX3 may modulate phagocytic functions of microglia, and this may have important implications in the regulation of microglial activity in health and disease.

Diverse functional roles of lipocalin-2 in the central nervous system

Lipocalin-2 (LCN2) is an acute phase protein with multiple functions that has garnered a great deal of interest over the last decade. However, its precise role in the pathophysiology of the central nervous system (CNS) remains to be outlined. Emerging evidence indicates that LCN2 is synthesized and secreted as an inducible factor from activated microglia, reactive astrocytes, neurons, and endothelial cells in response to inflammatory, infectious, or injurious insults. More recently, it has been recognized as a modulatory factor for diverse cellular phenotypes in the CNS, such as cell death, survival, morphology, migration, invasion, differentiation, and functional polarization. LCN2 induces chemokine production in the CNS in response to inflammatory challenges, and actively participates in the innate immune response, cellular influx of iron, and regulation of neuroinflammation and neurodegeneration. LCN2 also modulates several biobehavioral responses including pain hypersensitivity, cognitive functions, emotional behaviors, depression, neuronal excitability, and anxiety. This review covers recent advances in our knowledge regarding functional roles of LCN2 in the CNS, and discusses how LCN2 acts as an autocrine mediator of astrocytosis, a chemokine inducer, and a modulator of various cellular phenotypes in the CNS. We finally explore the possibilities and challenges of employing LCN2 as a signature of several CNS anomalies.

Role of soluble CD14 in cerebrospinal fluid as a regulator of glial functions.

Proteomic analysis of cerebrospinal fluid (CSF) samples derived from patients with Alzheimers disease (AD) or Parkinsons disease (PD) was performed. On the basis of liquid chromatography–tandem mass spectrometry, two‐dimensional gel electrophoresis analysis, and Western blot validation, it was found that the level of soluble form of monocyte differentiation antigen CD14 precursor was elevated in CSF from AD or PD patients compared with normal subjects. The soluble CD14 protein and mRNA expression was detected in microglia cells, indicating that microglia may be a cellular source of soluble CD14 in CSF. Next, the role of soluble CD14 in the regulation of glial functions was investigated. Soluble CD14 inhibited lipopolysaccharide (LPS)‐ or LPS/interferon‐gamma‐induced nitric oxide production and cell death of microglia and astrocytes. Soluble CD14 suppressed glial neurotoxicity in a coculture of glia/neuroblastoma. In addition, soluble CD14 moderately enhanced phagocytic activity of microglia. These results suggest that microglia‐derived soluble CD14 is a candidate CSF biomarker for AD and PD, and the soluble CD14 may inhibit glial activation by interfering with LPS effects.