MicroRNA-124 promotes microglia quiescence and suppresses EAE by deactivating macrophages via the C/EBP-α–PU.1 pathway

In the paper

“It has previously been shown that the TNF receptor (TNFR1) is a direct downstream target for C/EBP-α and C/EBP-β35”

What the sources say

• Supported

  The Introduction reports that both C/EBPα and C/EBPβ bind and activate the TNFR1 promoter, and that reducing their expression decreases TNFR1 protein expression. The Discussion explicitly states that both factors directly bind and activate the promoter driving the TNFR1 gene, supporting the characterization of TNFR1 as a direct downstream target. (LLM, conf=0.94)

  cites [35] Bristol, J.A., Morrison, T.E. & Kenney, S.C. CCAAT/enhancer binding proteins α and β regulate the tumor necrosis factor receptor 1 gene promoter. Mol. Immunol. 46, 2706–2713 (2009). source

In the paper

“In addition, miR-124 inhibits proliferation and induces differentiation of medulloblastoma and glioblastoma cancer cells, which originate from immature neural cells43,44.”

What the sources say

• ?Not checked — no full text of the sourcecites [43] Pierson, J., Hostager, B., Fan, R. & Vibhakar, R. Regulation of cyclin dependent kinase 6 by microRNA 124 in medulloblastoma. J. Neurooncol. 90, 1–7 (2008). source
• Partly supported

  The Conclusion and Discussion report that miR-124 promotes differentiation of glioblastoma-derived stem cells and inhibits proliferation of glioblastoma cells. Our copy does not report these effects in medulloblastoma cells, so this source supports only the glioblastoma portion of the claim. The supplied passages have no page numbers. (LLM, conf=0.88)

  cites [44] Silber, J. et al. miR-124 and miR-137 inhibit proliferation of glioblastoma multiforme cells and induce differentiation of brain tumor stem cells. BMC Med. 6, 14 (2008). sourceExperimental — accuracy not yet measured

In the paper

“and that TNF-α and TNFR1 may enhance each other's expression by an autocrine-paracrince mechanism36,37.”

What the sources say

• Partly supported

  The Introduction says that TNF-α first downregulates and then upregulates the 55-kD TNF receptor on human keratinocytes. The Discussion (p. 467) describes TNF-α acting through that receptor in an autocrine mechanism, but does not show that the receptor enhances TNF-α expression. The proposed mutual enhancement is therefore only partly supported. (LLM, conf=0.87)

  cites [36] Trefzer, U. et al. The 55-kD tumor necrosis factor receptor on human keratinocytes is regulated by tumor necrosis factor-α and by ultraviolet B radiation. J. Clin. Invest. 92, 462–470 (1993). sourceExperimental — accuracy not yet measured
• Not checked yetcites [37] Cardinaux, J.R., Allaman, I. & Magistretti, P.J. Pro-inflammatory cytokines induce the transcription factors C/EBPβ and C/EBPδ in astrocytes. Glia 29, 91–97 (2000). source

In the paper

“It promotes differentiation of neuronal progenitors to mature neurons by targeting small C-terminal domain phosphatase-1 and polypyrimidine tract–binding protein-1 that repress expression of neuronal genes in non-neuronal cells18,20,40.”

What the sources say

• Supported

  The Introduction (page unavailable) says miR-124 promotes neuronal differentiation in part by targeting PTBP1 and SCP1, identifying their roles in neuron-specific splicing and the REST repressor complex. The Discussion (page unavailable) also reports that this paper found miR-124 promotes neuronal differentiation in the adult subventricular zone. The targeting mechanism is presented as prior work, not as a mechanism newly established by this study. (LLM, conf=0.86)

  cites [18] Cheng, L.C., Pastrana, E., Tavazoie, M. & Doetsch, F. miR-124 regulates adult neurogenesis in the subventricular zone stem cell niche. Nat. Neurosci. 12, 399–408 (2009). source
• ?Not checked — no full text of the sourcecites [20] Makeyev, E.V., Zhang, J., Carrasco, M.A. & Maniatis, T. The MicroRNA miR-124 promotes neuronal differentiation by triggering brain-specific alternative pre-mRNA splicing. Mol. Cell 27, 435–448 (2007). source
• Partly supported

  The sections on neuronal differentiation in the developing spinal cord and SCP1-mediated neurogenesis in P19 cells report that miR-124 promotes neurogenesis by directly targeting SCP1. The passages do not identify polypyrimidine tract-binding protein-1 as a target, so this source supports only part of the attributed mechanism. Page numbers are unavailable in our copy. (LLM, conf=0.88)

  cites [40] Visvanathan, J., Lee, S., Lee, B., Lee, J.W. & Lee, S.K. The microRNA miR-124 antagonizes the anti-neural REST/SCP1 pathway during embryonic CNS development. Genes Dev. 21, 744–749 (2007). sourceExperimental — accuracy not yet measured

In the paper

“miR-124 is known to be expressed in the CNS by neuronal cells23,39–41.”

What the sources say

• ?Not checked — no full text of the sourcecites [23] Smirnova, L. et al. Regulation of miRNA expression during neural cell specification. Eur. J. Neurosci. 21, 1469–1477 (2005). source
• ?Not checked — no full text of the sourcecites [39] Mishima, T., Mizuguchi, Y., Kawahigashi, Y. & Takizawa, T. RT-PCR–based analysis of microRNA (miR-1 and -124) expression in mouse CNS. Brain Res. 1131, 37–43 (2007). source
• Supported

  The section “SCP1 is down-regulated by miR-124” describes the lateral neuronal domain of the developing spinal cord as a site where miR-124 is highly expressed. The Abstract also describes miR-124 as abundant in the adult and embryonic CNS. (LLM, conf=0.92)

  cites [40] Visvanathan, J., Lee, S., Lee, B., Lee, J.W. & Lee, S.K. The microRNA miR-124 antagonizes the anti-neural REST/SCP1 pathway during embryonic CNS development. Genes Dev. 21, 744–749 (2007). source
• Supported

  The Results section reports miR-124 expression in virtually all differentiating cells of the larval zebrafish brain and in most differentiated cells of the adult brain. It explicitly describes the adult-brain expression as occurring in neurons, supporting the claim that neuronal cells express miR-124 in the CNS. Page numbers were not provided in our copy. (LLM, conf=0.93)

  cites [41] Kapsimali, M. et al. MicroRNAs show a wide diversity of expression profiles in the developing and mature central nervous system. Genome Biol. 8, R173 (2007). source

In the paper

“These results are consistent with our observations that quiescent microglia show properties of M2 macrophages and that M2 macrophages are crucial for the suppression of EAE45.”

What the sources say

• Supported

  The Results section on microglial IL-4 and Ym1 expression says CNS microglia exhibit properties of alternatively activated macrophages, including under normal conditions. The Introduction states that the IL-4-dependent alternatively activated phenotype is required to control CNS inflammation and that EAE was more severe when CNS IL-4 production or signaling was deficient. (LLM, conf=0.88)

  cites [45] Ponomarev, E.D., Maresz, K., Tan, Y. & Dittel, B.N. CNS-derived interleukin-4 is essential for the regulation of autoimmune inflammation and induces a state of alternative activation in microglial cells. J. Neurosci. 27, 10714–10721 (2007). source

In the paper

“whereas myeloid-specific miR-223 (refs. 6,22) was expressed in the macrophages in the periphery, but not in the microglia (Fig. 1b).”

What the sources say

• ?Not checked — no full text of the sourcecites [6] Johnnidis, J.B. et al. Regulation of progenitor cell proliferation and granulocyte function by microRNA-223. Nature 451, 1125–1129 (2008). source
• Supported

  The citation is attached to “myeloid-specific miR-223,” not to the macrophage–microglia comparison, which the citing sentence assigns to its own Fig. 1b. The source’s Discussion states that miR-223 expression is restricted to the myeloid compartment; it does not establish the peripheral macrophage versus microglia finding. (LLM, conf=0.91)

  cites [22] Fukao, T. et al. An evolutionarily conserved mechanism for microRNA-223 expression revealed by microRNA gene profiling. Cell 129, 617–631 (2007). source

In the paper

“Moreover, PU.1 controls proliferation of macrophages by upregulating the receptor for M-CSF34.”

What the sources say

• Partly supported

  The Results show that increasing PU.1 raises macrophage surface M-CSF receptor expression and enhances M-CSF-dependent proliferation. But the paper calls the receptor a possible proliferation-relevant target; it does not establish that receptor upregulation is the mechanism by which PU.1 controls proliferation. Section and page labels are unavailable in our copy. (LLM, conf=0.87)

  cites [34] Celada, A. et al. The transcription factor PU.1 is involved in macrophage proliferation. J. Exp. Med. 184, 61–69 (1996). sourceExperimental — accuracy not yet measured

In the paper

“During onset of EAE, ~40% of peripheral macrophages showed a partially deactivated MHC class II− phenotype, whereas during recovery, ~40% of peripheral macrophages became CD45lowMHC class II− (Fig. 2a).”

In the paper

“During recovery, the level of MHC class II in microglia returned to normal, but a subset of cells remained CD45hi, suggesting some residual level of activation.”

In the paper

“In our experiments, activated microglia downregulated miR-124, and high levels of C/EBP-α expression were associated with the activated CD45hi phenotype.”

In the paper

“Figure 5d shows how the treatment with miR-124 decreased the absolute numbers of lymphocytes, CD4 T cells and macrophages and activated CD45hi microglia in the CNS.”

In the paper

“Mutations of this reporter construct within two predicted miR-124 binding sites (C/EBP-αmut1–2) substantially reduced the luciferase responsiveness to miR-124, and mutations within all three predicted sites (C/EBP-αmut1–3) nearly abolished it (Fig. 4e).”

In the paper

“Intravenously injected small interfering RNA (siRNA) specific for C/EBP-α had similar effects on disease symptoms, whereas mutant miR-124 lacking the C/EBP-α mRNA binding sequence had no effect on the disease score (Supplementary Data and Supplementary Fig. 9a).”

In the paper

“This conditional knockout reduced levels of C/EBP-α protein, which resulted in reduced expression of PU.1, CD11b, MHC class II and CD86 (Supplementary Data and Supplementary Fig. 7).”

In the paper

“We found that brain-specific miR-124 is expressed in microglia but not in peripheral monocytes or macrophages.”

In the paper

“Inflammatory lesions containing nucleated and CD11b+ cells in the white matter of the lumbar spinal cord were evident in the control group but undetectable in the miR-124–treated mice (Fig. 5e).”

In the paper

“When injected into C57BL/6→Cx3cr1GFP/+ chimeric mice that express GFP in microglia but not in peripheral macrophages, anti–miR-124 induced the activation of CD11b+GFP+ microglia and altered their morphology (Supplementary Data and Supplementary Fig. 16b–e).”

In the paper

“Quantification of the four experiments shows that miR-124 overexpression decreased the proportion of CD45hi C/EBP-α+ and CD45hiPU.1+ cells by 70% and 80%, respectively (Fig. 4d).”

In the paper

“Activated MHC class II+ peripheral macrophages expressed very low levels of miR-124, whereas deactivated MHC class II− macrophages upregulated miR-124 by 4.2-fold (Fig. 2b).”

In the paper

“During mouse prenatal and postnatal development, microglia showed an activated phenotype with a low level of miR-124 expression (Supplementary Data and Supplementary Fig. 3).”

In the paper

“Transfection of macrophages with miR-124 also inhibited expression of TNF-α (Fig. 3c) and inducible nitric oxide synthase (Fig. 3d) by these cells, and upregulated both the anti-inflammatory cytokine TGF-β1 (Fig. 3d,e) and markers of alternatively activated (M2) macrophages arginase I and FIZZ1 (Fig. 3d).”

In the paper

“Transfection with miR-124 also resulted in a reduced number of proliferating cells and altered cell morphology (Supplementary Data and Supplementary Fig. 4).”

In the paper

“Disease symptoms were also substantially ameliorated in both EAE-prone SJL mice as well as in Ifng−/− mice, which have a more severe form of EAE (Supplementary Data and Supplementary Fig. 8a,b).”

In the paper

“Two-color flow cytometry further confirmed that transfection of macrophages with miR-124 resulted in downregulation of both CD45 and C/EBP-α (Fig. 4c), and indicated that the C/EBP-α− phenotype corresponded to the CD45low phenotype of macrophages.”

In the paper

“In contrast, the mice treated with miR-124 had primarily CD11b+CD45low resting microglia (Fig. 5c, miR-124, region R1) and no signs of microglia activation or leukocyte infiltration in the CNS (Fig. 5c, miR-124, regions R2 and R3).”

In the paper

“Treatment of mice with miR-124 at the onset of EAE (starting day 13 after EAE induction) also substantially ameliorated clinical symptoms and enhanced recovery (Fig. 5b).”

In the paper

“Subsequent experiments showed that peripheral administration of miR-124 during EAE directly affected macrophages and deactivated them, and indirectly reduced the activation of autoimmune CD4 T cells (Supplementary Data, Supplementary Figs. 10–15 and Supplementary Table 3).”

In the paper

“C/EBP-α expression was reduced in miR-124–transfected BMDMs by 50–70% on the protein level (Fig. 4b) and by 50% on the mRNA level (Fig. 3d).”

In the paper

“The highest level of miR-124 expression was observed in resting CD45lowMHC class II− microglia, whereas activated MHC class II+ microglia expressed a 60% lower level of miR-124 (Fig. 2b).”

In the paper

“These data suggest that miR-124 not only deactivates macrophages but also skews their polarization from an M1 toward an M2 phenotype.”

In the paper

“In this study, we refer to cells expressing CD11b+F4/80+ as ‘macrophages’ even though they may include small numbers of CD11b+CD11c+F4/80+ myeloid dendritic cells. Peripheral macrophages may also be contaminated by small numbers of GFP+ microglia that enter the CNS during reconstitution14.”

What the sources say

• ?Not checked — no full text of the sourcecites [14] Ponomarev, E.D., Shriver, L.P., Maresz, K. & Dittel, B.N. Microglial cell activation and proliferation precedes the onset of CNS autoimmunity. J. Neurosci. Res. 81, 374–389 (2005). source

In the paper

“Our extensive analysis showed that in vivo administration of miR-124 directly affected macrophages, and this, in turn, resulted in deficient activation of encephalitogenic T cells (Supplementary Discussion).”

In the paper

“These data suggest that miR-124 controls multiple markers of macrophage activation by direct inhibition of C/EBP-α and its downstream transcription factor PU.1.”

In the paper

“These data confirm that miR-124 directly targets the mRNA encoding C/EBP-α and reduces expression of this protein.”

In the paper

“Thus, treatment of mice with miR-124 ameliorated both EAE symptoms and inflammation in the CNS.”

In the paper

“It has been shown with human HeLa cells and synoviocytes that miR-124 can regulate expression of multiple target genes, including those encoding CDKs and MCP-1 (refs. 17,42).”

What the sources say

• ?Not checked — no full text of the sourcecites [17] Lim, L.P. et al. Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs. Nature 433, 769–773 (2005). source
• ?Not checked — no full text of the sourcecites [42] Nakamachi, Y. et al. MicroRNA-124a is a key regulator of proliferation and monocyte chemoattractant protein 1 secretion in fibroblast-like synoviocytes from patients with rheumatoid arthritis. Arthritis Rheum. 60, 1294–1304 (2009). source

In the paper

“We have demonstrated that miR-124 directly inhibits expression of the C/EBP-α transcription factor, which results in downregulation of PU.1, another master regulator of monocytic cell differentiation and decreases expression of the macrophage activation markers CD45, CD11b, F4/80, MHC class II and CD86.”

In the paper

“Normal adult microglia do not express C/EBP-α, but this transcription factor can be induced during inflammation38.”

What the sources say

• ?Not checked — no full text of the sourcecites [38] Walton, M. et al. CCAAT-enhancer binding proteinα is expressed in activated microglial cells after brain injury. Brain Res. Mol. Brain Res. 61, 11–22 (1998). source

In the paper

“In summary, our findings demonstrate that miR-124 has a key role in inhibiting macrophage activation and promoting microglial differentiation under physiological conditions in the CNS microenvironment.”

In the paper

“These data demonstrate that miR-124 has a role in maintaining the quiescent phenotype of microglia in the normal CNS, and this phenotype is driven by the paracrine interaction of microglia with local stromal cells.”