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. 2017 Oct;18(10):1801-1816.
doi: 10.15252/embr.201643668. Epub 2017 Aug 14.

LncRNA GAS5 inhibits microglial M2 polarization and exacerbates demyelination

Dingya Sun  1 , Zhongwang Yu  1 , Xue Fang  1 , Mingdong Liu  1 , Yingyan Pu  1 , Qi Shao  1 , Dan Wang  1 , Xiaolin Zhao  1 , Aijun Huang  1 , Zhenghua Xiang  1 , Chao Zhao  2 , Robin Jm Franklin  2 , Li Cao  3 , Cheng He  3
Affiliations

LncRNA GAS5 inhibits microglial M2 polarization and exacerbates demyelination

Dingya Sun et al. EMBO Rep. 2017 Oct.

Abstract

The regulation of inflammation is pivotal for preventing the development or reoccurrence of multiple sclerosis (MS). A biased ratio of high-M1 versus low-M2 polarized microglia is a major pathological feature of MS Here, using microarray screening, we identify the long noncoding RNA (lncRNA) GAS5 as an epigenetic regulator of microglial polarization. Gain- and loss-of-function studies reveal that GAS5 suppresses microglial M2 polarization. Interference with GAS5 in transplanted microglia attenuates the progression of experimental autoimmune encephalomyelitis (EAE) and promotes remyelination in a lysolecithin-induced demyelination model. In agreement, higher levels of GAS5 are found in amoeboid-shaped microglia in MS patients. Further, functional studies demonstrate that GAS5 suppresses transcription of TRF4, a key factor controlling M2 macrophage polarization, by recruiting the polycomb repressive complex 2 (PRC2), thereby inhibiting M2 polarization. Thus, GAS5 may be a promising target for the treatment of demyelinating diseases.

Keywords: GAS5; M1/M2 polarization; demyelination; microglia; multiple sclerosis.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1. GAS5 is downregulated in M2‐polarized microglia

  1. Hierarchical clustering analysis of 120 lncRNAs that were differentially expressed between microglia treated with IL‐4 for 24 h and the untreated control (greater than twofold; P < 0.05). Expression values are represented in shades of red and green and indicate expression above and below the median expression value across all samples (log scale 2, from 3 to −3), respectively.

  2. Co‐expression network in IL‐4‐stimulated microglia. The co‐expression network includes 1,627 connections among 319 genes that are correlated at |r| > 0.99. The color of the nodes is in accordance with the K‐core number shown in the legend. Real lines between two nodes indicate positively correlated interactions between genes, and dashed lines indicate negatively correlated interactions. The enlarged box shows that GAS5 is a key in the network.

  3. Quantitative PCR analysis of GAS5 in microglia treated with IL‐4 for 24 h versus the untreated control, n = 3 experiments.

  4. Quantitative PCR analysis of GAS5 in M‐CSF‐treated microglia versus the untreated control, n = 3 experiments.

  5. Quantitative PCR analysis of GAS5 in rapamycin‐treated microglia versus the untreated control, n = 3 experiments.

  6. Quantitative PCR analysis of GAS5 in LY294002‐treated microglia versus the untreated control, n = 3 experiments.

Data information: *P < 0.05, **P < 0.01, ***P < 0.001 (Student's t‐test). Data are shown as the mean ± SD.
Figure 2
Figure 2. GAS5 suppresses microglial M2 polarization and promotes M1 polarization
  1. A, B

    Quantitative PCR analysis of M1 and M2 markers in microglia transduced with the GAS5OE (A) or GAS5i (B) lentivirus vectors versus the control, n ≥ 3 experiments.

  2. C, D

    ELISA analysis of TNF‐α, IL‐1β, and IGF‐1 in culture supernatants of microglia transduced with the GAS5OE (C) or GAS5i (D) lentivirus vectors versus the control, n ≥ 3 experiments.

  3. E, F

    Western blotting analysis of TNF‐α in microglia transduced with the GAS5OE (E) or GAS5i (F) lentivirus vectors versus the control, n ≥ 3 experiments.

Data information: *P < 0.05, ***P < 0.001 versus control (Student's t‐test). Data are shown as the mean ± SD.Source data are available online for this figure.
Figure EV1
Figure EV1. GAS5‐modified microglia affect OPC survival
  1. A, B

    Representative flow cytometry analysis after Annexin‐V/7‐AAD staining in OPCs incubated with conditioned medium from MGGAS5OE (A) or MGGAS5i (B) for 24 h versus the control.

  2. C, D

    Apoptosis rate of the OPCs in (C) and (D) respectively, n = 3 experiments.

  3. E, F

    TUNEL analysis of apoptosis in OPCs incubated with conditioned medium from MGGAS5OE (E) or MGGAS5i (F) for 24 h versus the control, n = 3 experiments. The ratio of TUNEL+/Hoechst+ cells was calculated and compared between groups. Scale bars = 50 μm.

Data information: *P < 0.05, **P < 0.01, ***P < 0.001 versus control (Student's t‐test). Data are shown as the mean ± SD.
Figure EV2
Figure EV2. GAS5‐modified microglia affect OPC differentiation and neurite growth
  1. A, B

    Anti‐MBP immunocytofluorescence of OPCs incubated with conditioned medium from MGGAS5OE (A) or MGGAS5i (B) for 48 h versus the control. Scale bars = 50 μm. The ratio of MBP+/Hoechst+ cells was calculated and compared between groups, n = 3 experiments.

  2. C, D

    Western blotting analysis of MBP in OPCs incubated with conditioned medium from MGGAS5OE (C) or MGGAS5i (D) for 48 h versus the control, n = 3 experiments.

  3. E

    Anti‐MAP2 immunocytofluorescence of neurons incubated with conditioned medium from MGGAS5OE or MGGAS5i for 48 h versus the control, n ≥ 4 experiments. Scale bar = 50 μm.

Data information: *P < 0.05, ***P < 0.001 versus control (Student's t‐test). Data are shown as the mean ± SD.Source data are available online for this figure.
Figure 3
Figure 3. GAS5 is dynamically expressed in the microglia of EAE
  1. A

    Representative FISH analysis of GAS5 (green) co‐stained with an anti‐Iba1 antibody (red) in spinal cord sections from EAE mice. Arrows indicate GAS5+Iba1+ cells. Scale bars = 100 μm.

  2. B

    A schematic map of the calculation of the ramification index (RI).

  3. C

    Statistical analysis of the RI in Iba1+GAS5+ (n = 16) or Iba1+GAS5− (n = 17) cells.

  4. D, E

    Representative FISH analysis of GAS5 (green) co‐stained with an anti‐TNF‐α (D) or anti‐Arg‐1 (E) antibody (red) in spinal cord sections from EAE mice. Arrows indicate GAS5+TNF‐α+ cells in (D) and GAS5+Arg‐1+ cells in (E). Scale bars = 25 μm.

  5. F

    Statistical analysis of the proportion of GAS5+ cells in (D, E), n ≥ 3 experiments.

  6. G

    A schematic map of the sorting of microglia in different phases of EAE and the control by FACS.

  7. H

    Quantitative PCR analysis of GAS5 in different phases of EAE versus the control, n = 3 experiments.

Data information: *P < 0.05, ***P < 0.001 (C, Student's t‐test; H, one‐way ANOVA with Dunnett's post hoc test). Data are shown as the mean ± SD.
Figure EV3
Figure EV3. GAS5 is expressed in the microglia of EAE
Representative FISH analysis of GAS5 (green) co‐stained with an anti‐TMEM119 antibody (red) in spinal cord sections from EAE mice at 30 dpi. Arrows indicate GAS5+TMEM119+ cells. Scale bars = 25 μm.
Figure 4
Figure 4. GAS5‐modified microglia regulate the progression of EAE
  1. A, B

    Clinical scores of EAE in mice transplanted with MGCtrlOE/MGGAS5OE (A) or MGCtrli/MGGAS5i (B) by lateral ventricle injection. CtrlOE, n = 18; GAS5OE, n = 18; Ctrli, n = 8; GAS5i, n = 10 mice.

  2. C, D

    Representative spinal cord sections from EAE mice at day 20 after immunization after H&E staining (C) and Luxol fast blue staining (D). Scale bars = 100 μm.

  3. E, F

    Quantification of infiltration in the white matter (WM) (E) and the percentage of demyelinated WM in total WM (F) in (C and D), n ≥ 3 mice per group.

Data information: *P < 0.05, **P < 0.01, ***P < 0.001 versus control (A, B, Mann–Whitney U‐test; E, F, Student's t‐test). Data are shown as the mean ± SEM (A, B) or mean ± SD (E, F).
Figure 5
Figure 5. Interfering with GAS5 in microglia attenuates EAE progression
  1. A

    Clinical scores of EAE in mice transplanted with MGCtrli/MGGAS5i by lateral ventricle injection, n = 6 mice per group.

  2. B, C

    Representative spinal cord sections of H&E stained (B) and Luxol fast blue stained (C) EAE mice day 40 after immunization. Scale bars = 100 μm.

  3. D, E

    Quantification of infiltration in the white matter (WM) (D) and the percentage of demyelinated WM in total WM (E) in (B and C), n = 3 mice per group.

Data information: *P < 0.05, **P < 0.01, ***P < 0.001 versus control (A, Mann–Whitney U‐test; D, E, Student's t‐test). Data are shown as the mean ± SEM (A) or mean ± SD (D, E).
Figure 6
Figure 6. Interfering with GAS5 in microglia promotes remyelination in a LPC‐induced focal lesion model
  1. A

    Representative Luxol fast blue‐stained dorsal column spinal cord sections from the MGCtrli/MGGAS5i groups at 7, 11, and 15 days post‐LPC injection. Scale bar = 100 μm.

  2. B

    Quantitative analysis of the demyelinated lesion volume 5 mm around the epicenter of (A), n = 3 mice per group.

  3. C

    Representative anti‐Olig2 (green) and anti‐CC1 (red) immunohistofluorescence of dorsal column spinal cord sections from the MGCtrli/MGGAS5i groups at 7, 11, and 15 days post‐LPC injection. Scale bar = 100 μm.

  4. D, E

    Statistical analysis of Olig2+ (D) or CC1+ (E) cells of (C), n ≥ 3 mice per group.

  5. F

    Representative electron micrographs of dorsal column spinal cord sections from the MGCtrli/MGGAS5i groups at 15 days post‐LPC injection. Scale bar = 2 μm.

  6. G

    Quantification analysis of myelinated axons among total axons between MGCtrli and MGGAS5i groups at 15 dpl, n = 3 mice per group.

  7. H

    Statistical analysis of g‐ratio between MGCtrli and MGGAS5i groups at 15 dpl.

  8. i

    Analysis of the myelinated axons showed a reduction of the g‐ratio in spinal cords in the MGGAS5i group (red) compared with the MGCtrli group (blue) at 15 dpl, n = 3 mice per group.

Data information: **P < 0.01, ***P < 0.001 versus control (Student's t‐test). Data are shown as mean ± SD.
Figure EV4
Figure EV4. Analysis of the mechanism regulating microglial polarization by GAS5
  1. A

    Silver staining to identify specific binding partners of GAS5 after RNA pull‐down experiment.

  2. B

    RNA IP analysis between GR and GAS5. N = 3 experiments.

  3. C

    RNA IP analysis between RbAp48 and GAS5. N = 3 experiments.

  4. D

    RNA IP analysis between RING1A/B and GAS5. N = 3 experiments.

  5. E–G

    ChIP analysis of microglia transduced with the CtrlOE or GAS5OE lentivirus. The promoter regions of STAT6 (E), PPARγ (F) and CEBP/β (G) were detected in MGGAS5OE versus the control using the anti‐EZH2 antibody.

Data information: ***P < 0.001 (B–D, Student's t‐test). Data are shown as the mean ± SD.
Figure 7
Figure 7. GAS5 suppresses IRF4 transcription by binding PRC2 and inhibiting microglial M2 polarization
  1. A

    RNA IP analysis of the binding between EZH2 and GAS5, n = 3 experiments.

  2. B

    RNA pull‐down analysis of the binding between EZH2 and GAS5.

  3. C

    Quantitative PCR analysis of M1 and M2 markers in microglia transduced with the EZH2i lentivirus versus the control, n ≥ 4 experiments.

  4. D

    ChIP analysis of microglia transduced with the CtrlOE or GAS5OE lentivirus. A relatively high enrichment was detected on the IRF4 promoter regions in MGGAS5OE versus the control using the anti‐EZH2 antibody, n = 3 experiments.

  5. E

    ChIRP analysis of the binding between the IRF4 promoter and GAS5, n = 3 experiments. IGF1 served as a negative control.

  6. F, G

    ChIP analysis of microglia transduced with the CtrlOE/GAS5OE (F) or Ctrli/GAS5i (G) lentivirus on the IRF4 promoter regions using the anti‐H3K27me3 antibody, n = 3 experiments.

  7. H–J

    Quantitative PCR analysis of IRF4 in microglia transduced with the GAS5OE (H), GAS5i (I) or EZH2i (J) lentivirus, n ≥ 3 experiments per group.

  8. K–M

    Western blotting analysis of IRF4 in microglia transduced with the GAS5OE (K), GAS5i (L), or EZH2i (M) lentivirus, n ≥ 3 experiments per group.

Data information: *P < 0.05, **P < 0.01, ***P < 0.001 versus control or IgG (A) (Student's t‐test). Data are shown as the mean ± SD.Source data are available online for this figure.
Figure 8
Figure 8. GAS5 inhibits human microglial M2 polarization and is differentially expressed in MS lesions

  1. Quantitative PCR analysis of M1 and M2 markers in human microglia nucleo‐transfected with the human GAS5 plasmid versus the control, n ≥ 3 experiments.

  2. Quantitative PCR analysis of M1 and M2 markers in mouse microglia nucleo‐transfected with the human GAS5 plasmid versus the control, n = 3 experiments.

  3. Representative FISH analysis of GAS5 (green) co‐stained with the anti‐Iba1 antibody (red) in brain slices from MS patients. Arrows indicate GAS5+Iba1+ cells. Scale bar = 50 μm.

  4. Statistical analysis of the RI of Iba1+GAS5+ or Iba1+GAS5− cells in brain slices from MS patients, n = 6.

Data information: *P < 0.05, **P < 0.01, ***P < 0.001 (Student's t‐test). Data are shown as the mean ± SD.
Figure EV5
Figure EV5. GAS5 is highly expressed in aged microglia and regulates the inflammatory response of monocytes
  1. A

    A schematic map of microglial isolation from young (6 weeks) and aged mice (> 15 months).

  2. B

    Quantitative PCR analysis of GAS5 in microglia from aged mice versus young mice, n = 3 experiments.

  3. C, D

    Quantitative PCR analysis of M1 and M2 markers in monocytes transduced with the GAS5OE (C) or GAS5i (D) lentivirus vectors versus the control, n ≥ 3 experiments.

Data information: *P < 0.05, **P < 0.01, ***P < 0.001 (B–D, Student's t‐test). Data are shown as the mean ± SD.
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