Immune Netw.  2011 Dec;11(6):342-347. 10.4110/in.2011.11.6.342.

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

• Kim H
• Lee S
• Park HC
• Lee WH
• Lee MS
• Suk K

• KMID: 2150718
• DOI: http://doi.org/10.4110/in.2011.11.6.342

Abstract

BACKGROUND
Glial 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.
METHODS
Here, 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.
RESULTS
Conditioned 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.
CONCLUSION
Thus, LCN2 proteins secreted under diverse conditions may play an important role in CNS immune and inflammatory responses by controlling cell migration and morphology.

Keyword

LCN2; Glia; Cell migration; Neuroinflammation; Central nervous system; Spinal cord

MeSH Terms

Astrocytes
Cell Movement
Central Nervous System
Culture Media, Conditioned
Embryonic Structures
Microglia
Neuroglia
Neurons
Proteins
Spinal Cord
Zebrafish
Culture Media, Conditioned
Proteins

Figure

• 

  Figure 1 Regulation of glial and neuronal migration by LCN2. Microglia, astrocytes, or neuronal cells (1×104 cells/upper well) were exposed to control (CTRL) or LCN2 (10µg/ml)-stimulated astrocyte-conditioned media (ACM) as indicated. Microglia, astrocytes, or neuronal cells placed in the Boyden chambers were incubated at 37℃ for 48 hr to evaluate cell migration. CTRL-ACM, untreated ACM; LCN2-ACM, LCN2-treated ACM (see Materials and Methods for the preparation of ACM). The quantification of cell migration was done by enumerating the migrated cells as described in the Materials and Methods section. The results are mean±SD (n=3). *p<0.05 compared with CTRL-ACM.

• 

  Figure 2 The effect of LCN2-treated astrocyte conditioned media (LCN2-ACM) on the morphology of cortical neuron cells. ACM was prepared after the treatment of primary astrocytes with LCN2 (10µg/ml) or LPS (100 ng/ml) plus IFN-γ (50 units/ml) for 24 hr. The addition of LCN2-treated ACM (LCN2-ACM) induced morphological changes in primary cortical neuron cells after 24 hr. LPS (100 ng/ml) plus IFN-γ (50 units/ml)-treated ACM (LPS/IFN-γ-ACM), which was used for comparison, also induced similar morphological changes (A). Primary cortical neuron cells were stained with MAP2 antibody (magnification, ×100) followed by the incubation with anti-mouse IgG-fluorescein isothiocyanate (FITC)-conjugated secondary antibody (scale bar, 25µm). The insets indicate the magnified images. The results are one representative of more than three independent experiments. The total number of neuronal process was counted for each field (B). The results are mean±SD (n=3). *p<0.05; compared with the untreated ACM control (CTRL-ACM).

• 

  Figure 3 Ectopic expression of LCN2 promotes neuronal migration in the spinal cord of zebrafish embryos. The her4:egfp-injected control embryo (A) or her4:lcn2:egfp-injected embryo (B) was labeled with an anti-Hu antibody to detect neurons at 24 hpf. Arrowheads indicate neurons near the egfp-expressing cells (A) or lcn2:egfp-expressing cells (B). Dotted lines indicate midline of the spinal cord. All images are transverse sections of zebrafish spinal cord, dorsal to top.

• 

  Figure 4 The expression of LCN2 attracts developing neurons toward medial position of the spinal cord in zebrafish. The wild-type embryo (A) or her4:lcn2:egfp-injected transgenic embryo (B) was labeled with an anti-Hu antibody to detect neurons at 24 hpf. Arrowheads indicate neurons near the lcn2:egfp-expressing cells. Dotted lines indicate a lateral margin (a) and medial position (b) of the spinal cord. Numbers indicate percentage of neuronal cells in each region. All images are transverse sections of zebrafish spinal cord, dorsal to top. The quantification of cell migration was done by enumerating the migrated cells as described in the Materials and Methods section. The results are mean±SD.

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