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Keywords:

  • brain injury;
  • inflammation;
  • interleukin-6;
  • neuronal death;
  • reactive astrogliosis

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments and conflict of interest disclosure
  7. References
  8. Supporting Information
Thumbnail image of graphical abstract

N-myc downstream-regulated gene 2 (Ndrg2) is a differentiation- and stress-associated molecule predominantly expressed in astrocytes in the CNS. In this study, we examined the expression and the role of Ndrg2 after cortical stab injury. We observed that Ndrg2 expression was elevated in astrocytes surrounding the wounded area as early as day 1 after injury in wild-type mice. Deletion of Ndrg2 resulted in lower induction of reactive astroglial and microglial markers in the injured cortex. Histological analysis showed reduced levels of hypertrophic changes in astrocytes, accumulation of microglia, and neuronal death in Ndrg2−/− mice after injury. Furthermore, activation of the IL-6/signal transducer and activator of transcription 3 (STAT3) pathway, including the expression of IL-6 family cytokines and phosphorylation of STAT3, was markedly reduced in Ndrg2−/− mice after injury. In a culture system, both of Il6 and Gfap were up-regulated in wild-type astrocytes treated with forskolin. Deletion of Ndrg2 attenuated induction of these genes, but did not alter proliferation or migration of astrocytes. Adenovirus-mediated reexpression of Ndrg2 rescued the reduction of IL-6 expression after forskolin stimulation. These findings suggest that Ndrg2 plays a key role in reactive astrogliosis after cortical stab injury through a mechanism involving the positive regulation of IL-6/STAT3 signaling.

Astrocytes play a key role in central nervous system (CNS) injury. We demonstrate that Ndrg2 (N-myc downstream-regulated gene 2) is up-regulated in the early phase after brain injury, and that its deletion alters astrogliosis, inflammation, and neuronal death in vivo. The function of Ndrg2 could correlate with the interleukin IL-6 pathway in astrocytes. Our findings have important implications for the modulation of inflammation in the CNS.

Abbreviations used
DAPI

6-diamidino-2-phenylindole dihydrochloride

Fsk

forskolin

GFAP

glial fibrillary acidic protein

IL-6

interleukin-6

Ndrg2

N-myc downstream-regulated gene 2

NeuN

neuronal nuclei

qRT-PCR

quantitative real-time PCR

ssDNA

single-strand DNA

STAT3

signal transducer and activator of transcription 3

Astrocytes, which are the most abundant cells in the CNS, play pivotal roles not only in the nutritional support for neurons but also in the regulation of neural activity at the tripartite synapse by modulation of extracellular transmitters and at sites of neurovascular coupling by controlling microcirculation (Araque et al. 1999; Iadecola and Nedergaard 2007; Schummers et al. 2008). In addition to these homeostatic functions in physiological conditions, astrocytes play important roles in various neuropathological conditions, including acute brain insults, such as trauma and stroke, and chronic neurodegeneration. In these conditions, astrocytes undergo hypertrophy and up-regulate intermediate filament proteins such as glial fibrillary acidic protein (GFAP) and vimentin, which are referred to as reactive astrogliosis or astroglial activation. In some cases, astrocytes undergo proliferation and form a tight barrier between the injury site and the healthy areas, termed a glial scar (Sofroniew 2009). Reactive astrogliosis has both detrimental and beneficial aspects. Astrocytes are known to release inflammatory mediators and participate in innate immune reactions, and the glial scar inhibits axonal regeneration after injury (Silver and Miller 2004; Brambilla et al. 2005; Widestrand et al. 2007). However, recent studies using mice with experimentally ablated astroglial function have demonstrated that activated astrocytes exert neuroprotection through the prevention of spread of inflammatory cells and restoration of tissue damage (Sung et al. 2003; Okada et al. 2006; Huang et al. 2012). Therefore, it is critical to understand the molecular mechanism underlying reactive astrogliosis to appropriately regulate its level and timing.

N-myc downstream-regulated gene 2 (Ndrg2) is one of the recently identified differentiation- and stress- associated genes, and a high level of Ndrg2 expression has been observed in the brain, heart, and muscle, and to a lesser extent in the liver and kidney (Qu et al. 2002). Ndrg2 is a cytoplasmic protein that contains several phosphorylation sites (Murray et al. 2004). Its structure is similar to that of the members of the alpha-beta hydrolase family, but it lacks the enzymatic sequence (Hwang et al. 2011); therefore, its direct function and the target proteins are still unknown. An increasing number of studies have indicated a correlation between down-regulation of Ndrg2 (Lusis et al. 2005; Jeschke et al. 2012) and increased levels of proliferation (Deng et al. 2003; Kim et al. 2009ab) and/or invasion (Kim et al. 2009a) of cancer cells. Recently, analysis using Ndrg2 gain-of-function and loss-of-function mutant mice showed that Ndrg2 regulates the vertebral specification in the differentiating somite (Zhu et al. 2012). In the CNS, Ndrg2 has been reported to be predominantly expressed in astrocytes (Nichols 2003, Okuda et al. 2008; Takeichi et al. 2011) and in dystrophic neurons in Alzheimer's disease (Mitchelmore et al. 2004). Ndrg2 has also been reported to be up-regulated by adrenal steroid hormones (Boulkroun et al. 2002; Nichols 2003) and also in a relatively early phase after experimental ischemia (Li et al. 2011), while being down-regulated by antidepressant and electroconvulsive treatments (Takahashi et al. 2005a). In our previous study, we demonstrated that Ndrg2 has an inhibitory effect on the proliferation of astrocytes in a culture system and that it is highly expressed in astrocyte-like cells in the substantia nigra of Parkinson's disease patients (Takeichi et al. 2011). These lines of evidence indicate that Ndrg2 expression in astrocytes could be involved in the pathogenesis of neurological disorders. In this study, we investigated the expression of Ndrg2 and the effects of its deletion on the processes of astroglial activation, inflammatory response, and neuronal death in a cortical stab injury model.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments and conflict of interest disclosure
  7. References
  8. Supporting Information

Mice

All animal experiments were approved by the Animal Care and Use Committee of Kanazawa University (AP-122395). Ndrg2-deficient mice were generated through a combination of homologous recombination and Cre-mediated recombination. The targeting vector, including two loxP sites flanking exons 3 and 4 of the mouse Ndrg2 locus and neomycin (Neo) cassette, was linearized and transfected into 129Sv/J embryonic stem cells by electroporation. Recombinant embryonic stem clones were selected with G418 antibiotic and identified by PCR analysis. Secondary confirmation was performed by Southern blotting analysis by using two types of [32P] labeled probes for the target region of genomic DNA after digestion with EcoT221 (5′ probe) or DraI (3′ probe). The expected sizes were 8.2 and 8.8 kb for the wild-type (WT) allele and 9.8 and 9.9 kb for the targeted allele. Two correctly targeted heterozygote embryonic stem cell clones were obtained and were microinjected into C57BL/6 blastocysts. The resulting chimeras were mated to C57BL/6 mice (Sankyo Labo Service Corporation, Tokyo, Japan) to generate F1 heterozygous offspring. Germ line transmission of Ndrg2 mutant gene was achieved in one of those lines. Ndrg2neo/+ mice were crossed with CAG-Cre transgenic mice that express Cre recombinase under a CAG promoter to generate Ndrg2+/− mice. CAG-Cre mice were kindly provided by Dr Miyazakai (Osaka university, Osaka, Japan) (Sasaki and Miyazaki 1997). Mice were backcrossed into the C57BL/6 background for eight generations. Genotyping was performed by PCR using tail genomic DNA. Primer sequences are listed in Table S1.

Cortical stab injury

Adult male mice aged 3–4 months were used for all the experiments. Cortical stab injury was performed as described earlier (Auguste et al. 2007; Buffo et al. 2008; Robel et al. 2011a), with slight modifications. Briefly, mice were anesthetized deeply with chloral hydrate at a dose of 400 mg/kg by intraperitoneal injection (Nacalai Tesque, Kyoto, Japan). A stab wound lesion was created with a scalpel blade by sagittal insertion through the skull to the right cerebral cortex at a depth of 1.0, 1.7 mm lateral to the midline, and 1.0–2.5 mm posterior to the bregma. The mice were killed from 1 to 7 days after the stab injury and subjected to the morphological and biochemical analyses described below.

Histological and immunohistochemical analyses

Brains were removed from the C57BL/6 mice after perfusion with 4% paraformaldehyde, and cortical sections (10-μm-thick sections) were cut on a cryostat. Sections were processed for Fluoro-Jade C staining (Millipore, Billerica, MA, USA) or for immunostaining with antibodies against Ndrg2 (Santa Cruz Biotechnology, Dallas, TX, USA), GFAP (Dako, Glostrup, Denmark; Sigma, St. Louis, MO, USA), Iba1 (Wako, Osaka, Japan), neuronal nuclei (Millipore), nestin (Millipore), SRY-box containing gene 2 (Sox2) (Santa Cruz Biotechnology), single-strand DNA (ssDNA) (Dako), and S100β (Sigma). Alexa488- (Invitrogen, Carlsbad, CA, USA) or Cy3-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) was used for visualization of immunolabeling. Imaging was performed on a laser scanning confocal microscope (Eclipse TE2000U; Nikon, Tokyo, Japan) with the Nikon EZ-C1 software. The immunoreactivity of GFAP was quantified using ImageJ (version 1.42, Wayne Rasband, National Institutes of Health, Bethesda, MD, USA), and the number of Iba1-positive cells was counted in four 0.07-mm2 rectangles stepwise from the center of the stab wound to a distance of 0.4 mm. The number of Fluoro-Jade C- or ssDNA-positive cells, and the number of GFAP- and interleukin-6 (IL-6) double-positive cells were counted in 0.27-mm2 rectangles at the center of the lesion.

Western blot and ELISA

Samples from mouse brains, including the stab wound lesion or the corresponding region in the contralateral side, and from cultured astrocytes were solubilized in buffer containing 10 mM Tris (pH 7.6), 1 mM EDTA, 150 mM NaCl, 1% NP-40, 0.1% sodium dodecyl sulfate, 0.2% sodium deoxycholate, 1 mM phenylmethylsulfonyl fluoride, 1 μg/mL aprotinin, 10 mM NaF, and 1 mM Na3VO4. Western blot was performed using antibodies against Ndrg2, GFAP, HO-1 (Abcam, Cambridge, UK), phospho-signal transducer, and activator of transcription 3 (STAT3) (Tyr705) (Cell Signaling Technology, Danvers, MA, USA), STAT3 (Cell Signaling Technology), and β-actin (Sigma). Sites of primary antibody binding were determined by using an enhanced chemiluminescence system (GE Healthcare, Pittsburgh, PA, USA) with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology). The intensity of each band was quantified using ImageJ (version 1.42). The IL-6 levels in the brain samples and the culture media of astrocytes were measured using an ELISA kit (eBioscience, San Diego, CA, USA) following the manufacturer's instructions.

Quantitative real-time RT-PCR

Total RNA was extracted from cultured astrocytes and the contralateral or ipsilateral cortex after stab injury by using the RNeasy Lipid Tissue Mini Kit (Qiagen, Valencia, CA, USA). cDNA was prepared using PrimeScript (Takara, Shiga, Japan) with 1 μg of total RNA. Each cDNA sample was amplified in a reaction mixture containing 1 unit of Taq DNA polymerase (Takara) and primers specific for Ndrg2. For quantitative analysis, cDNA was amplified with Thunderbird SYBR qPCR Mix (Toyobo, Osaka, Japan) by using primers specific for Gfap, Hmox1 (heme oxygenase 1), Serpina3n (serine peptidase inhibitor clade A member 3), Lcn2 (lipocalin 2), Iba1, Cxcl1 (chemokine [C-X-C motif] ligand 1), Ccl2 (chemokine [C-C motif] ligand 2), Tnfa (tumor necrosis factor α), Lif (leukemia-inhibitory factor), Il6 (interleukin-6), Cntf (ciliary neurotrophic factor), Tgfb1 (transforming growth factor β), Edn1 (endothelin-1), Ndrg2, Atf3 (activating transcription factor 3), and Ccnd1 (cyclin D1). The comparative Ct method was used for data analyses with MxPro 4.10 (Agilent Technologies, Santa Clara, CA, USA). The expression levels for each gene were normalized to those of Gapdh (glyceraldehyde-3-phosphate dehydrogenase) or Actb (β-actin). Primer sequences are listed in Table S1.

Recombinant adenovirus

Recombinant adenovirus AxCALNLZ2 Ndrg2 (AdV-Ndrg2) expressing FLAG (DYKDDDDK)-tagged Ndrg2 under the control of Cre-recombinase was developed as described before (Takeichi et al. 2011), and adenovirus was amplified in HEK293 cells. Viral titers were determined by the tissue culture infectious dose method (TCID50) in HEK293 cells. Viral stocks had titers of ~ 1 × 108 PFU/mL. Cre-recombinase-expressing adenovirus AxCANCre and the control virus AxCALNLZ2 LacZ (AdV-LacZ) were obtained from Takara.

Cell culture

Astrocytes were isolated from cerebral cortex of 1- to 3-day-old neonatal mice following a previously described method with minor modifications (Kuwabara et al. 1996). Briefly, cerebral hemispheres were harvested from neonatal mice, and the meninges were carefully removed. Brain tissue was then digested at 37°C in HEPES-buffered saline containing Dispase II (2 mg/mL). Cells were collected by centrifugation and resuspended in minimum essential medium supplemented with 10% fetal bovine serum. After 10-day cultivation, microglial cells were removed by aspiration after shaking and the adherent cell population was collected and used for experiments. When the cultures achieved confluence, cells were incubated in serum-free medium overnight and then treated with several astroglial activators such as forskolin (10 μM; Wako), ATP (1 mM; Sigma), lipopolysaccharide (1 μg/mL; Sigma), endothelin-1 (200 nM; Sigma), and TGFβ (25 ng/mL; Peprotech Inc. London, UK) in serum-free medium for indicated time. In some cases, cells were coinfected with Cre-expressing adenovirus and either Ndrg2-expressing adenovirus (AdV-Ndrg2) or a control adenovirus (AdV-LacZ), in which gene expression was controlled by the Cre-loxP system. Two days after adenovirus infection, the cells were incubated in the presence or absence of 10 μM forskolin for 8 h in serum-free medium. Total RNA and culture media were subjected to quantitative real-time RT-PCR (qRT-PCR) and ELISA, respectively.

3-(4, 5-Dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide assay

Cell proliferation analysis was performed by the 3-(4, 5-Dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay to assess the mitochondrial activity at every 24 h up to 5 days after plating. Astrocytes isolated from WT or Ndrg2−/− mice were incubated with 0.5 mg/mL MTT (Nacalai Tesque) for 1 h at 37°C. After aspiration of the MTT solution, the formazan crystals were dissolved in dimethyl sulfoxide. The absorbance of the dissolved solution was measured at a wavelength of 590 nm.

Scratch wound assay

Confluent monolayers of astrocytes were wounded by scratching with a sterile plastic 10 μL tip from left side to right side of the well (Robel et al. 2011a). Culture medium was immediately changed to remove the cell debris. Images were taken 24 h and immediately after medium change with using light microscope. The wound area was measured using ImageJ, as described in Histological and immunohistochemical analyses section.

Luciferase assay

Cultured astrocytes were cotransfected with cAMP-response element (CRE)-luciferase reporter plasmid (Stratagene) and control plasmid pRL-SV40 Renilla luciferase (Promega) using Lipofectamine 2000 (Invitrogen) as the transfection agent. Cells were incubated in serum-free medium overnight and then treated with forskolin at 10 μM for 6 h. The dual luciferase assay was performed according to the manufacturer's protocol (Promega). For each samples, the firefly luciferase activity (CRE-Luc) was normalized by reference to renilla luciferase activity (pRL-SV40).

Statistical analysis

All the results have been expressed in terms of mean ± SEM, and statistical significance was determined by two-tailed and unpaired Student's t-tests or one-way anova with the Bonferroni/Dunnett post hoc test.

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments and conflict of interest disclosure
  7. References
  8. Supporting Information

Elevated expression of Ndrg2 and GFAP in the injured cerebral cortex

Consistent with our previous report (Takeichi et al. 2011), Ndrg2 was expressed selectively in astrocytes in the normal cerebral cortex (Figure S1a). To investigate the expression of Ndrg2 in the course of reactive astrogliosis, we used a mouse cortical stab injury model, which is a well-established model of acute brain injury (Auguste et al. 2007; Buffo et al. 2008; Robel et al. 2011a). Immunohistochemical analysis revealed that expression of GFAP, which is a marker of reactive astrocytes, began to elevate at day 1 after injury in fine processes of astrocytes and further increased at day 4 after injury in hypertrophic astrocytes around the lesion (Fig. 1a). It was rarely seen in the contralateral cortex with the exception in the surface of region. Western blot analysis revealed that expression of Ndrg2 protein was significantly higher in the ipsilateral side than that in the contralateral side, as early as day 1 after injury, in which significant GFAP induction did not occur yet (Fig. 1b). Immunohistochemical analysis also indicated high levels of Ndrg2 expression in the ipsilateral side, compared with the contralateral side at day 1 after injury in the both GFAP-positive and GFAP-negative cells around the lesion (Fig. 1c and Figure S1b). At day 4, most of the Ndrg2-expressing cells were colabeled with GFAP, but not with neuronal nuclei or Iba1, the latter two are neuronal and microglial cell markers, respectively (Fig. 1d). Some of the Ndrg2-expressing cells in the injured cortex were colabeled with nestin (Fig. 1d), which is a marker for progenitor cells and also reported to be expressed in reactive astrocytes (Buffo et al. 2008; Robel et al. 2011b; Shimada et al. 2012). These data indicate that expression of Ndrg2 is selectively observed in astrocytes, and its enhancement precedes that of GFAP after cotrical stab injury.

image

Figure 1. Increased Ndrg2 expression in astrocytes after cortical stab injury. (a) Glial fibrillary acidic protein (GFAP) immunostaining was performed using brain sections obtained at day 1 and day 4 after cortical stab injury. The ipsilateral (ipsi) and the contralateral (contra) areas are presented. Dotted lines indicate the stab wounds. Enlarged picture indicates a single GFAP-positive astrocyte. Scale bars = 100 μm. (b) Ndrg2 and GFAP protein expression from day 1 to day 7 after cortical stab injury. Protein extracts from the lesion site (ipsi: ipsilateral) and control side (c: contralateral) were subjected to western blot analysis. Quantified data are presented in the right graph. Data are shown as mean ± SEM (n = 3–5). *p < 0.05, **p < 0.01 versus the contralateral cortex. (c) Ndrg2 (red) and GFAP (green) immunostaining was performed using brain sections obtained at day 1 after injury. The ipsilateral and the contralateral areas are presented. High magnification of framed area is shown in the enlarged picture. Dotted lines indicate the stab wounds. Scale bars = 50 μm. (d) Immunostaining for Ndrg2 (red) and cellular markers (green) including GFAP, Iba1, neuronal nuclei (NeuN) and nestin, was performed using brain sections obtained at day 4 after injury. Scale bars = 50 μm.

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Generation of Ndrg2-deficient mice

To investigate the role of Ndrg2 in vivo, we generated Ndrg2 knockout mouse using homologous recombination and the Cre/loxP system. As shown in Fig. 2a, Ndrg2 was targeted with loxP-flanking sequences containing exons 3 and 4 of Ndrg2. Successful targeting was confirmed by Southern blot analysis (Fig. 2b). Then, Ndrg2neo/+ mice were crossed with CAG-Cre transgenic mice to generate Ndrg2+/− mice. Ndrg2−/− mice grew normally and were fertile. Disruption of Ndrg2 was confirmed in several tissues such as the brain, liver, and heart at both mRNA and protein levels by RT-PCR and western blot analysis, respectively (Fig. 2c and d).

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Figure 2. Targeted disruption of Ndrg2. (a) Schematic representation of the wild-type (WT) Ndrg2 locus, targeting vector, targeted allele, and deleted allele. The targeting vector replaces the coding region of exons 3 and 4 with loxP-flanked exons 3 and 4. Neomycin resistance cassette was removed in the cAMP-response element (CRE)-deleted allele. (b) Southern blot-based analysis of genomic DNA isolated from the WT ES cells (+/+) and the Ndrg2-targeted ES cells (+/neo). Targeted allele was confirmed by two different probes that recognize the 5′ site and 3′ site of fragments digested with EcoT221 and DraI, respectively. (c) PCR analysis and (d) western blot analysis showing deletion of Ndrg2 in the brain, liver, and heart from mice homozygous for the Ndrg2-deleted allele.

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Impaired glial response in Ndrg2-deficient mice after stab injury

To study the effect of Ndrg2 deletion after brain injury, WT and Ndrg2−/− mice received a cortical stab injury. In WT mice, the stab injury markedly increased the mRNA expression of the following genes, which started to increase at day 1 and peaked at day 4 after injury (Fig. 3a and Figure S2a): reactive astroglial markers such as Gfap, Serpna3n, and Lcn2 (Naudé et al. 2012; Zamanian et al. 2012); the microglial marker Iba1; the anti-oxidative gene Hmox1 known as HO-1 (Park et al. 2008; Hashida et al. 2012); and inflammatory mediators such as Cxcl1, Ccl2, and Tnfa. In Ndrg2−/− mice, the levels of mRNA expression of these genes were significantly lower than those of WT mice at day 1 and/or day 4 after stab injury (Fig. 3a). Consistent with these results, the levels of protein expression of GFAP, Iba1, and HO-1 were significantly lower in Ndrg2−/− mice at day 4 after stab injury (Fig. 3b and Figure S2e). Similarly, immunohistochemical analysis showed that the intensity of the GFAP signal was significantly lower in Ndrg2−/− mice than in WT mice in all the regions within 400 μm from the injury site (Fig. 3c). In contrast, the number of Iba1-positive microglia was significantly lower in Ndrg2−/− mice in the regions closer to the injury site (Fig. 3c). Taken together with the fact that Ndrg2 is expressed in astrocytes, reduced levels of microglial accumulation in Ndrg2−/− mice after stab injury may be a secondary effect of impaired astroglial activation.

image

Figure 3. Glial response after cortical stab injury in Ndrg2-deficient mice (a) temporal gene expression profile for gliosis markers and chemokines after stab injury. Total RNA prepared from the ipsilateral (ipsi) and contralateral (c) cortices of wild-type (WT) mice and Ndrg2−/− mice from day 1 to day 7 after injury were subjected to quantitative RT-PCR using primers specific for Gfap, Hmox1, Serpina3n, Lcn2, Iba1, and Ccl2. Data are shown as mean ± SEM (n = 3–4). *p < 0.05, **p < 0.01 versus each contralateral cortex. #p < 0.05, ##p < 0.01 versus indicated conditions. (b) Protein expression of glial fibrillary acidic protein (GFAP) and HO-1 from day 1 to day 7 after cortical stab injury. Protein extracts from the ipsilateral (ipsi) and contralateral (c) cortices of WT mice and Ndrg2−/− mice were used for western blot analysis. Quantified data are presented in the right graphs. Data are shown as mean ± SEM (n = 3–5). #p < 0.05 versus indicated conditions. (c) Brain sections were prepared from WT mice and Ndrg2−/− mice at day 4 after cortical stab injury and subjected to immunostaining for GFAP (red) and Iba1 (green). Lower panels represent high magnification of the white box in the upper panels. Enlarged picture in lower panels indicates a representative single GFAP-positive astrocyte. Dotted lines indicate the stab wound. Scale bars = 200 μm (upper panel), 50 μm (lower panel). Quantified intensity of GFAP immunostaining and cell numbers of Iba1-positive cells are presented in the right graphs. Data are shown as mean ± SEM (n = 6). #p < 0.05, ##p < 0.01 versus indicated conditions.

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Impaired IL-6/STAT3 signaling in Ndrg2-deficient mice

To investigate whether impaired astroglial activation in Ndrg2−/−mice occurs at early periods after stab injury, the expression of several astroglial activators such as IL-6 family cytokines, including Il6, Lif and Cntf, and other soluble factors such as Tgfβ and Edn1 were analyzed by qRT-PCR. Cortical stab injury strongly induced Il6 and Lif at day 1 and Cntf at day 4 after stab injury in WT mice, but the induction was significantly reduced in Ndrg2−/− mice (Fig. 4a). In our model, Tgfb1 or Edn1 was not significantly induced in both genotypes (Figure S2b). Consistent with these results, immunohistochemical analysis revealed that IL-6-positive astrocytes were observed more often in the lesion site of WT mice than Ndrg2−/− mice at day 1 after injury (Fig. 4b). In addition, there was a tendency for decrease in the level of IL-6 protein, which was examined by ELISA; the level was lower in Ndrg2−/− mice than in WT mice after stab injury (Figure S2d; 37.4 pg/mL in the ipsilateral side and 16.8 pg/mL in the contralateral side in WT mice, and 24.4 pg/mL in the ipsilateral side and 17.1 pg/mL in the contralateral side in Ndrg2−/− mice). Next, the status of IL-6 signaling was studied. The binding of IL-6 family cytokines to their receptors triggers the activation of STAT3, which has been reported to play a critical role in astrogliosis (Okada et al. 2006; Herrmann et al. 2008). Cortical stab injury induced phosphorylation of STAT3 at tyrosine 705 in WT mice to a level significantly higher than that in Ndrg2−/− mice (Fig. 4c).

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Figure 4. Interleukin-6 (IL-6)/signal transducer and activator of transcription 3 (STAT3) signaling in Ndrg2-deficient mice. (a) Temporal gene expression profile for astroglial activators after stab injury. Total RNA was extracted from ipsilateral and contralateral cortices from day 1 to day 7 after injury and subjected to qRT-PCR using primers specific for Il6, Lif, and Cntf. Data are shown as mean ± SEM (n = 3–4). *p < 0.05 versus indicated conditions. (b) Brain sections were prepared at day 1 after stab injury and subjected to immunostaining for glial fibrillary acidic protein (GFAP) (green) and IL-6 (red). GFAP and IL-6 double-positive cells in the white box are enlarged in the lower panels. Scale bars = 20 μm (upper panel), 10 μm (lower panel). Quantified data are presented in the right graph. Data are shown as mean ± SEM (n = 5). *p < 0.05 versus wild-type (WT) mice. (c) Phosphorylation of STAT3 at day 1 after stab injury. Protein extracts from the ipsilateral (ipsi) and contralateral (c) cortices of WT and Ndrg2−/− mice were subjected to western blot analysis with the indicated antibodies. Quantified data are presented in the right graph. Data are shown as mean ± SEM (n = 5) **p < 0.01 versus WT mice.

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Attenuated response in Ndrg2-deficient astrocytes after forskolin stimulation

To examine whether the impaired glial response in Ndrg2−/− mice was the effect of Ndrg2 deficiency in astrocytes, we employed primary astroglial cultures derived from WT and Ndrg2−/− mice. In cultured astrocytes, there was no difference in proliferation and migration between both genotypes determined by MTT reduction assay and scratch wound assay, respectively (Fig. 5a and b). In the latter assay in WT astrocytes, there was no significant difference in the expression of Ndrg2, Il6, or Gfap (Figure S3a and b). Next, astrocytes were stimulated by the several reagents known to induce astroglial activation in vitro and/or in vivo (Gadea et al. 2008; Sofroniew 2009; Schachtrup et al. 2010). Western blot analysis revealed that the expression of Ndrg2 was enhanced most by forskolin treatment among the reagents tested (Fig. 5c, d and e). Consistent with the results in vivo, the level of induction of Il6 and Gfap was significantly lower in Ndrg2−/− astrocytes 8 h and 24 h after forskolin treatment, respectively, although the CRE reporter activity was somehow increased at 6 h (Fig. 5f, g and h). To investigate whether down-regulation of IL-6 expression was caused by the deletion of Ndrg2, reexpression of Ndrg2 in Ndrg2−/− astrocytes was carried out by the infection of Ndrg2-expressing adenovirus. The expression levels of Ndrg2 were determined by qRT-PCR and western blot (Fig. 5i and j). The reduced level of IL-6 expression in Ndrg2−/− astrocytes was completely restored by adenovirus-mediated Ndrg2 expression at both mRNA and protein levels (Fig. 5k and l), while the restoration in Gfap expression was milder (Figure S3c). These results suggest that the Ndrg2 positively regulates IL-6 expression in astrocytes.

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Figure 5. Response of Ndrg2-deficient astrocytes in vitro. (a) Cell proliferation of wild-type (WT) or Ndrg2−/− astrocytes was analyzed by the 3-(4, 5-Dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide (MTT) reduction assay at every 24 h up to day 5. Data are shown as mean ± SEM (n = 4). (b) The scratch wound assay was performed to evaluate cell migration by using cultured astrocytes isolated from WT or Ndrg2−/− mice. The quantification of cell migration was done by the measurement of coverage of the wound area at 24 h after scratching. Data are shown as mean ± SEM (n = 3). (C-E) Protein expression of Ndrg2 and glial fibrillary acidic protein (GFAP) in cultured astrocytes. Cells were stimulated with the indicated reagents for 48 h, and protein extracts were subjected to western blot analysis (c). The protein expression levels of Ndrg2 (d) and GFAP (e) under forskolin treatment were quantified. Data are shown as mean ± SEM (n = 4). *p < 0.05 versus control. Fsk: Forskolin, LPS: Lipopolysaccharide, ET1: Endothelin-1, TGFβ: Transforming growth factor-β. (f–h) Cultured astrocytes isolated from WT or Ndrg2−/− mice were stimulated by 10 μM forskolin. cAMP-response element (CRE) reporter activity was determined by dual luciferase assay in astrocytes isolated from WT or Ndrg2−/− mice after the cultivation for 6 h in the presence or absence of 10 μM forskolin (f). Gene expression levels were determined after 10 μM forskolin treatment for Il6 (g) at 8 h and Gfap (h) at 24 h by qRT-PCR. **p < 0.05 versus astrocytes without forskolin treatment. ##p < 0.01 versus indicated conditions. (i–l) Astrocytes isolated from WT or Ndrg2−/− mice were infected with the Ndrg2 adenovirus or LacZ adenovirus and cultured for 8 h in the presence or absence of 10 μM forskolin. Ndrg2 expression in Ndrg2−/− astrocytes was determined after adenovirus infection by qRT-PCR (I) and western blot (j). Il6 expression in the cells and interleukin-6 (IL-6) secretion levels in cultured media were determined by qRT-PCR (k) and ELISA (l), respectively. Data are shown as mean ± SEM (n = 5–8). The values of IL-6 mRNA and protein in WT astrocytes stimulated with forskolin after infection of LacZ adenovirus were designated as hundred. #p < 0.05, ##p < 0.01 versus indicated conditions.

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Attenuated neuronal death in Ndrg2−/− mice after cortical stab injury

To evaluate the neuronal damage in Ndrg2−/− mice after stab injury, histological and immunohistochemical analyses were performed at day 4 after lesion. Both Fluoro-Jade C staining and ssDNA immunostaining, which detect apoptotic cells and degenerating neurons, respectively, revealed that Ndrg2−/− mice exhibited significantly less damage in the wounded area than WT mice (Fig. 6a). Most of the cells positive for ssDNA in their nuclei were also positive for Fluoro-Jade C in both genotypes (WT: 97.5%, Ndrg2−/− mice: 97.8%). Consistent with this finding, qRT-PCR revealed that the induction level of Atf3, which is known to increase in response to neuronal injury (van der Weerd et al. 2010; Zhang et al. 2011), was significantly reduced in Ndrg2−/− mice (Fig. 6b). These results indicated that Ndrg2 deletion leads to reduction of neuronal death after cortical stab injury.

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Figure 6. Attenuated neuronal damage in Ndrg2-deficient mice after cortical stab injury. (a) Brain sections prepared from wild-type (WT) and Ndrg2−/− mice at day 4 after cortical stab injury were subjected to staining with FluoroJade C (green) or immunostaining with the anti-ssDNA antibody (red). Scale bars = 50 μm. Quantified data are presented in the right graphs. Data are shown as mean ± SEM (n = 5). *p < 0.05 versus WT mice. (b) Temporal gene expression profile for neuronal injury marker. The total RNA extracted from the ipsilateral and contralateral cortices of WT and Ndrg2−/− mice at days 1–7 after injury was subjected to qRT-PCR with primers specific for Atf3. Data are shown as mean ± SEM (n = 3–4). *p < 0.05 versus indicated conditions.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments and conflict of interest disclosure
  7. References
  8. Supporting Information

In this study, we demonstrated that Ndrg2 plays an important role in the positive regulation of early reactive astrogliosis, at least in part, through the activation of IL-6/STAT3 signaling after cortical stab injury. In addition, the deletion of Ndrg2 resulted in the attenuated recruitment of microglia to the lesion site and prevented neuronal death. To our knowledge, this is the first report clarifying the role of Ndrg2 in pathological conditions in vivo through the use of Ndrg2-deficient mice.

Reactive astrogliosis is not an all-or-none response but is a graded spectrum of molecular changes associated with cellular hypertrophy, proliferation, and glial scar formation (Sofroniew and Vinters 2010). It is accompanied by the up-regulation of intermediate filament proteins including GFAP. Important finding in this study is that Ndrg2 is an early injury-responsive gene in astrocytes. Up-regulation of Ndrg2 was observed at day 1 after injury, in which that of GFAP was partially observed by immunohistochemical analysis (Figs 1a–c and 3b). Furthermore, we demonstrated that the induction of reactive astroglial markers, such as GFAP, Serpina3n, and Lcn2 (Fig. 3a and b), and cellular hypertrophy (Fig. 3c) were significantly lower in Ndrg2−/− mice than in WT mice after cortical stab injury. These observations are consistent with our previous report describing the role of Ndrg2 in the morphological changes in cultured astrocytes (Takeichi et al. 2011). In contrast, the effect of Ndrg2 on cell proliferation during astrogliosis was not clear in this study. Previous studies by us and other groups demonstrated the inhibitory role of Ndrg2 on cell proliferation in different tumor cells and in astrocytes (Deng et al. 2003; Kim et al. 2009a,b; Takeichi et al. 2011). There may be a couple of explanations for this discrepancy. One is the functional redundancy of Ndrg2 in the astroglial proliferation. In most of the previous reports, Ndrg2 was transiently silenced at mRNA level (Foletta et al. 2009) or inactivated at the protein level by over-expressing mutant proteins (Kim et al. 2009a,b), while Ndrg2 was genetically deleted from the developmental/embryonic stages in this study. This may cause altered gene expression by complementary mechanisms. Another explanation is that the cortical stab injury used in this study was relatively mild and may not cause high levels of astroglial proliferation. In this regard, the induction of Ccnd1, a gene that encodes CyclinD1, a marker of cell cycle progression in G1/S phase, was very mild (less than 2-fold increase in both genotypes) after stab injury (Figure S2c).

Among the triggering signals for astrogliosis that were investigated in this study, IL-6 was the most active molecule after stab injury. Activation of IL-6/STAT3 signaling in the injured CNS has been reported to induce strong astrogliosis. Loss of STAT3 or suppressor of cytokine signaling 3, a negative feedback molecule of STAT3, in mice under the control of GFAP or the nestin promoter has been reported to reduce or enhance reactive astrogliosis, respectively, in a spinal cord injury model (Okada et al. 2006; Herrmann et al. 2008). In this study, the effect of Ndrg2 deletion on IL-6 expression was observed to a larger extent in mouse brains (Fig. 4a) than in cultured astrocytes (Fig. 5g), suggesting that Ndrg2 may promote IL-6 expression through both a cell-autonomous mechanism in astrocytes and a non-cell-autonomous mechanism through other type of cells including microglia. Consistent with this, not only reactive astrogliosis but also microglial accumulation was impaired in Ndrg2−/− mice after stab injury (Fig. 3a and c), as previously reported in Il6−/− mice after cortical freeze injury (Penkowa et al. 2000). Ndrg2 may contribute to a positive feedback loop for IL-6 signaling, which is referred to as IL-6 amplifier activation (Murakami and Hirano 2012).

Although Il6−/−, GFAP-Stat3−/−, Nestin-Stat3−/−, and Ndrg2−/− mice demonstrated similar phenotypes in reactive astrogliosis, they demonstrated different phenotypes in neurons. Neuronal damage was exaggerated in the first three types of mice (Penkowa et al. 2000; Okada et al. 2006; Herrmann et al. 2008), while it was attenuated in Ndrg2−/− mice (Fig. 6). This discrepancy could be attributed to the difference of the severity of the insult or to the inflammatory response in each pathological model. In this regard, it is noteworthy that the expression level of chemokines and activation of microglia was reduced in Ndrg2−/− mice. Furthermore, induction of Lcn2 (lipocalin-2), a chemokine inducer released from reactive astrocytes and promotes neuronal death (Bi et al. 2013), was reduced after stab injury in Ndrg2−/− mice (Fig. 3a). Although it is still controversial whether astrogliosis is beneficial or detrimental, Ndrg2−/− mice will be a useful tool for studying the effect of glial activation on the neuronal survival/activity in different disease models.

It has been reported that Ndrg2 is up-regulated by adrenal steroid hormones (Nichols 2003), which occurs through the glucocorticoid receptor but not through the glucocorticoid response element half-site (GRE1/2) (Takahashi et al. 2005b). In the current cortical stab injury model, Ndrg2 might be up-regulated through the stress-associated steroid hormones or some stress-associated signals, including protein kinase A, as described in cultured astrocytes in this study (Fig. 5c and d). Further studies are required to help clarify the molecular mechanism underlying the regulation of Ndrg2 expression.

In conclusion, this study demonstrated that Ndrg2 is an injury-responsive gene that is involved in the early phase of astroglial activation and that it positively regulates reactive astrogliosis and the subsequent inflammatory response, at least in part, through the activation of IL-6/STAT3 signaling. Thus, Ndrg2 could be a new therapeutic target in various neurological conditions related to astroglial activation and inflammation.

Acknowledgments and conflict of interest disclosure

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments and conflict of interest disclosure
  7. References
  8. Supporting Information

The authors declare no conflicts of interest. This work was supported by a Grant-in-Aid for young scientists (B) (23790219) from Japanese Society for the Promotion of Science. We thank Ms Yoko Esaki and Ms Kiyo Kawata (NPO Biotechnology Research and Development) for their efforts in the generation of Ndrg2 chimeric mice. The CAG-Cre mice were kindly provided by Dr Junichi Miyazaki (Osaka University). We are grateful to Ms Nahoko Okitani, Mr Takashi Tamatani, and Ms Michiyo Tamatani for technical assistance.

All experiments were conducted in compliance with the ARRIVE guidelines.

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  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments and conflict of interest disclosure
  7. References
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments and conflict of interest disclosure
  7. References
  8. Supporting Information
FilenameFormatSizeDescription
jnc12729-sup-0001-FigS1-S3-TableS1.pdfapplication/PDF9629K

Figure S1. Ndrg2 expression in the normal cerebral cortex and validation of Ndrg2 antibody.

Figure S2. Gene expression after cortical stab injury.

Figure S3. Gene expression in cultured astrocytes.

Table S1. Primer sequences used for RT-PCR analysis.

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