CoChat
Sign Up
Cited by 237

The DOI matches its Crossref record; 2 of the 2 references it cites were found in a registry or an index.

Toll-like receptor 8 functions as a negative regulator of neurite outgrowth and inducer of neuronal apoptosis

Ma, Yinghua, Li, Jianxue, Chiu, Isaac, Wang, Yawen, Sloane, Jacob A., Lü, Jining, Kosaras, Bela, Sidman, Richard L., Volpe, Joseph J., Vartanian, Timothy·2006·The Journal of Cell Biology
DOI 10.1083/jcb.200606016PMC PMC2064562
Open this paper in CoChat — free account

Abstract

Toll receptors in Drosophila melanogaster function in morphogenesis and host defense. Mammalian orthologues of Toll, the Toll-like receptors (TLRs), have been studied extensively for their essential functions in controlling innate and adaptive immune responses. We report that TLR8 is dynamically expressed during mouse brain development and localizes to neurons and axons. Agonist stimulation of TLR8 in cultured cortical neurons causes inhibition of neurite outgrowth and induces apoptosis in a dissociable manner. Our evidence indicates that such TLR8-mediated neuronal responses do not involve the canonical TLR–NF-κB signaling pathway. These findings reveal novel functions for TLR8 in the mammalian nervous system that are distinct from the classical role of TLRs in immunity.

Overview

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Integrity signals

Registry

Crossref · 10.1083/jcb.200606016

Cites

2 of 2 references verified0 citation errors · 0 not checkable

Statistics

Figures

Figure 1. TLR8 is dynamically expressed during mouse brain development and localizes to axons and neurons. (A) Western blot analysis of TLR8 expression in the developing mouse brains. Spleen (Sp) and Raw264.7 (Raw) macrophages are positive controls for anti-TLR8 immunoreactivity. β-actin serves as loading control. (B) Immunohistochemical analysis of TLR8 expression in sagittal sections of E12 embryo, E18 brain, and P14 cerebral cortex. The images of the E12 embryo and E18 brain were acquired by confocal microscopy using the Tile Scan function. (C) Whole-mount in situ hybridization of E12 embryo using a digoxin (DIG)-labeled probe specific to Tlr8 mRNA. Arrowheads in B and C indicate the sympathetic nerve trunk. (D) Western blotting of TLR4, TLR7, TLR8, MyD88, and NF-κB subunit p65 in cortical neurons cultured for 1 (DIV1) and 5 d (DIV5). (E) Immunocytochemistry of TLR8 in cultured cortical neurons. MAP2 and neurofilament 200 kD (NFL) are neuron-specific markers. An affinity-purified anti-TLR8 polyclonal antibody was used in A, B, D, and E. P, postnatal day; P12w, 12-wk-old; ic, internal capsule; IMZ, cortical intermediate zone; f1, fimbria of hippocampus, OC, optic chiasm, ONL, olfactory nerve layer. Bars: (B, top, and C) 1 mm; (B, bottom) 100 μm; (E) 50 μm.
Figure 1. TLR8 is dynamically expressed during mouse brain development and localizes to axons and neurons. (A) Western blot analysis of TLR8 expression in the developing mouse brains. Spleen (Sp) and Raw264.7 (Raw) macrophages are positive controls for anti-TLR8 immunoreactivity. β-actin serves as loading control. (B) Immunohistochemical analysis of TLR8 expression in sagittal sections of E12 embryo, E18 brain, and P14 cerebral cortex. The images of the E12 embryo and E18 brain were acquired by confocal microscopy using the Tile Scan function. (C) Whole-mount in situ hybridization of E12 embryo using a digoxin (DIG)-labeled probe specific to Tlr8 mRNA. Arrowheads in B and C indicate the sympathetic nerve trunk. (D) Western blotting of TLR4, TLR7, TLR8, MyD88, and NF-κB subunit p65 in cortical neurons cultured for 1 (DIV1) and 5 d (DIV5). (E) Immunocytochemistry of TLR8 in cultured cortical neurons. MAP2 and neurofilament 200 kD (NFL) are neuron-specific markers. An affinity-purified anti-TLR8 polyclonal antibody was used in A, B, D, and E. P, postnatal day; P12w, 12-wk-old; ic, internal capsule; IMZ, cortical intermediate zone; f1, fimbria of hippocampus, OC, optic chiasm, ONL, olfactory nerve layer. Bars: (B, top, and C) 1 mm; (B, bottom) 100 μm; (E) 50 μm.CC BY-NC-SA · PubMed Central
Figure 2. R-848 inhibits neurite outgrowth and triggers apoptosis in freshly cultured cortical neurons. (A) Representative micrographs of neurons untreated (control) or treated with 500 μM loxoribine (Lox; TLR7 agonist), 100 μM R-848 (TLR7/8 agonist), or 5 μg/ml LPS (TLR4 agonist) for 24 h. Cells were fixed and double-immunostained with anti–βIII-tubulin (green) and anti-cleaved caspase3 (red) monoclonal antibodies. (B–D) Quantification of the effects of PAMPs on neurite length (B), neurite number (C), and cell death (D) as characterized by condensed nuclei (<50 ± 25 μm2). (E) Representative micrographs of R-848–treated cultures show a neuron (βIII-tubulin/TUJ1) with a condensed nucleus (DAPI; arrowhead) stained by anti-cleaved caspase3. Dose–response curve (F and G) and time-course (H and I) of R-848 effects on neurite outgrowth (F and H) and apoptosis (G and I). In F and G, neurons were treated for 24 h; in H and I, neurons were exposed to 100 μM R-848. (J and K) Quantification of neurite length (J) and apoptosis (K) of neurons cultured for 24 h in normal or conditioned medium with or without 100 μM R-848. In a separate culture, neurons grown in normal medium were first stimulated with 100 μM R-848 for 18 h, washed thoroughly to eliminate trace amounts of R-848, and further incubated in fresh normal medium for 12 h, after which the supernatant was collected as the conditioned medium. Statistical analysis was done by t test. *, P < 0.05; **, P < 0.01 versus controls (untreated, 0 μM or 0 h). Data are the mean ± the SEM for triplicate determinations. Bars: (A) 100 μm; (E) 50 μm.
Figure 2. R-848 inhibits neurite outgrowth and triggers apoptosis in freshly cultured cortical neurons. (A) Representative micrographs of neurons untreated (control) or treated with 500 μM loxoribine (Lox; TLR7 agonist), 100 μM R-848 (TLR7/8 agonist), or 5 μg/ml LPS (TLR4 agonist) for 24 h. Cells were fixed and double-immunostained with anti–βIII-tubulin (green) and anti-cleaved caspase3 (red) monoclonal antibodies. (B–D) Quantification of the effects of PAMPs on neurite length (B), neurite number (C), and cell death (D) as characterized by condensed nuclei (<50 ± 25 μm2). (E) Representative micrographs of R-848–treated cultures show a neuron (βIII-tubulin/TUJ1) with a condensed nucleus (DAPI; arrowhead) stained by anti-cleaved caspase3. Dose–response curve (F and G) and time-course (H and I) of R-848 effects on neurite outgrowth (F and H) and apoptosis (G and I). In F and G, neurons were treated for 24 h; in H and I, neurons were exposed to 100 μM R-848. (J and K) Quantification of neurite length (J) and apoptosis (K) of neurons cultured for 24 h in normal or conditioned medium with or without 100 μM R-848. In a separate culture, neurons grown in normal medium were first stimulated with 100 μM R-848 for 18 h, washed thoroughly to eliminate trace amounts of R-848, and further incubated in fresh normal medium for 12 h, after which the supernatant was collected as the conditioned medium. Statistical analysis was done by t test. *, P < 0.05; **, P < 0.01 versus controls (untreated, 0 μM or 0 h). Data are the mean ± the SEM for triplicate determinations. Bars: (A) 100 μm; (E) 50 μm.CC BY-NC-SA · PubMed Central
Figure 3. Neurite outgrowth inhibition and neuronal apoptosis induced by R-848 are dissociable. (A) Neurite length distribution of the cleaved caspase3-positive (C3+; n = 64) and the cleaved caspase3-negative neurons (C3−; n = 338) in cultures treated with 100 μM R-848 for 24 h. Cells with neurite length of <10 μm are defined as having no neurites, and cells with neurites longer than the average neurite length of the untreated neurons (56.4 ± 5.9 μm) are considered as having normal neurites (indicated by the horizontal bar). The insert shows a representative field of R-848–treated cultures. Asterisk and arrowheads indicate a C3+ cell with normal neurites and C3− cells with no neurites, respectively. Cells were fixed and double-immunostained with anti–βIII-tubulin (green) and anti-cleaved caspase3 (red) antibodies. (B) Representative micrographs of neurons untreated (control) or treated with 100 μM R-848 for 24 h in the presence of vehicle control (1% DMSO) or caspase3 inhibitor (20 μM Z-DEVD-FMK). (C and D) Quantification of the effects of blocking caspase3 activity on R-848–induced apoptosis (C) and neurite outgrowth inhibition (D). (E and F) Quantification of neurite length (E) and apoptosis (F) of neurons that were initially exposed to 100 μM R-848 for 12 h (the time when the R-848–induced morphological changes become detectable), then switched to R-848–free or –containing medium for a further incubation of 36 h. Control cultures were never exposed to R-848. Statistical analysis was done by t test. **, P < 0.01 versus controls. Data are the mean ± the SEM for triplicate determinations. Bars, 50 μm.
Figure 3. Neurite outgrowth inhibition and neuronal apoptosis induced by R-848 are dissociable. (A) Neurite length distribution of the cleaved caspase3-positive (C3+; n = 64) and the cleaved caspase3-negative neurons (C3−; n = 338) in cultures treated with 100 μM R-848 for 24 h. Cells with neurite length of <10 μm are defined as having no neurites, and cells with neurites longer than the average neurite length of the untreated neurons (56.4 ± 5.9 μm) are considered as having normal neurites (indicated by the horizontal bar). The insert shows a representative field of R-848–treated cultures. Asterisk and arrowheads indicate a C3+ cell with normal neurites and C3− cells with no neurites, respectively. Cells were fixed and double-immunostained with anti–βIII-tubulin (green) and anti-cleaved caspase3 (red) antibodies. (B) Representative micrographs of neurons untreated (control) or treated with 100 μM R-848 for 24 h in the presence of vehicle control (1% DMSO) or caspase3 inhibitor (20 μM Z-DEVD-FMK). (C and D) Quantification of the effects of blocking caspase3 activity on R-848–induced apoptosis (C) and neurite outgrowth inhibition (D). (E and F) Quantification of neurite length (E) and apoptosis (F) of neurons that were initially exposed to 100 μM R-848 for 12 h (the time when the R-848–induced morphological changes become detectable), then switched to R-848–free or –containing medium for a further incubation of 36 h. Control cultures were never exposed to R-848. Statistical analysis was done by t test. **, P < 0.01 versus controls. Data are the mean ± the SEM for triplicate determinations. Bars, 50 μm.CC BY-NC-SA · PubMed Central
Figure 4. R-848 effects on neurons are attenuated by a polyclonal antibody specifically against TLR8. (A) Representative micrographs of nonimmune IgG- and anti-TLR8 (α-TLR8)–transduced neurons that were either untreated (control) or treated with 100 μM R-848 for 18 h. Cells were fixed and double-immunostained with anti–βIII-tubulin (green) and anti-cleaved caspase3 (red) antibodies. Bar, 50 μm. (B and C) Quantification of the anti-TLR8–mediated blocking effects on R-848–induced neurite outgrowth inhibition (B) and apoptosis (C). Statistical analysis was done by t test. *, P < 0.05 versus nonimmune IgG-transduced neurons treated with R-848. Data are the mean ± the SEM for triplicate determinations.
Figure 4. R-848 effects on neurons are attenuated by a polyclonal antibody specifically against TLR8. (A) Representative micrographs of nonimmune IgG- and anti-TLR8 (α-TLR8)–transduced neurons that were either untreated (control) or treated with 100 μM R-848 for 18 h. Cells were fixed and double-immunostained with anti–βIII-tubulin (green) and anti-cleaved caspase3 (red) antibodies. Bar, 50 μm. (B and C) Quantification of the anti-TLR8–mediated blocking effects on R-848–induced neurite outgrowth inhibition (B) and apoptosis (C). Statistical analysis was done by t test. *, P < 0.05 versus nonimmune IgG-transduced neurons treated with R-848. Data are the mean ± the SEM for triplicate determinations.CC BY-NC-SA · PubMed Central
Figure 5. TLR8 stimulation in neurons does not activate the canonical TLR–NF-κB signaling pathway, but rather down-regulates IκBα and IRAK4. (A) ELISA assay for NF-κB (p65) transactivation using nuclear extracts from cortical neurons stimulated with 100 μM R-848, 500 μM loxoribine, 5 μg/ml LPS, or 10 ng/ml TNFα for the indicated times. LPS and TNFα serve as negative and positive controls, respectively. (B) Western blotting of the hallmarks of the conventional TLR-signaling pathway with lysates from neurons and Raw264.7 macrophages treated with 100 μM R-848 for the indicated times. (C) Quantification of changes in IκBα levels in R-848–stimulated neurons by band densitometry. A representative blot is shown in B. (D) Western blotting of IRAK4 in neurons stimulated with 100 μM R-848 for the indicated times. Note that TLR8 levels remain unchanged. (E) Quantification of changes in IRAK4 levels by band densitometry. A representative blot is shown in D. Data in C and E, expressed as percentage normalized to controls (100%), are the mean ± the SEM for pooled Western-blots from three independent cultures. Statistical analysis was done by t test. *, P < 0.05 versus controls.
Figure 5. TLR8 stimulation in neurons does not activate the canonical TLR–NF-κB signaling pathway, but rather down-regulates IκBα and IRAK4. (A) ELISA assay for NF-κB (p65) transactivation using nuclear extracts from cortical neurons stimulated with 100 μM R-848, 500 μM loxoribine, 5 μg/ml LPS, or 10 ng/ml TNFα for the indicated times. LPS and TNFα serve as negative and positive controls, respectively. (B) Western blotting of the hallmarks of the conventional TLR-signaling pathway with lysates from neurons and Raw264.7 macrophages treated with 100 μM R-848 for the indicated times. (C) Quantification of changes in IκBα levels in R-848–stimulated neurons by band densitometry. A representative blot is shown in B. (D) Western blotting of IRAK4 in neurons stimulated with 100 μM R-848 for the indicated times. Note that TLR8 levels remain unchanged. (E) Quantification of changes in IRAK4 levels by band densitometry. A representative blot is shown in D. Data in C and E, expressed as percentage normalized to controls (100%), are the mean ± the SEM for pooled Western-blots from three independent cultures. Statistical analysis was done by t test. *, P < 0.05 versus controls.CC BY-NC-SA · PubMed Central

Verification

Claims the paper makes

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

The self-check needs the paper’s full text.

Citations

Cites

Show all 2 references

Research works printed but not linked: 19

  1. 2004, Nat. Rev. Neurosci., 5, 521, 10.1038/nrn1428 · DOI · Paper page
  2. 2002, J. Biol. Chem., 277, 1991, 10.1074/jbc.M107992200 · DOI
  3. 2000, Eur. Cytokine Netw., 11, 362
  4. 2000, J. Neurosci., 20, 7556, 10.1523/JNEUROSCI.20-20-07556.2000 · DOI
  5. 2004, J. Leukoc. Biol., 76, 904, 10.1189/jlb.0504277 · DOI
  6. 2003, Eur. J. Immunol., 33, 2987, 10.1002/eji.200324238 · DOI
  7. 2003, Nature., 426, 33, 10.1038/nature02021 · DOI · Paper page
  8. 1999, Science., 284, 1313, 10.1126/science.284.5418.1313 · DOI
  9. 2002, Nat. Immunol., 3, 499, 10.1038/ni0602-499 · DOI · Paper page
  10. 2006, Nature., 440, 233, 10.1038/nature04517 · DOI
  11. 2006, J. Neurosci. Res., 83, 711, 10.1002/jnr.20767 · DOI
  12. 2003, Proc. Natl. Acad. Sci. USA., 100, 8514, 10.1073/pnas.1432609100 · DOI · Paper page
  13. 2005, Exp. Cell Res., 307, 15, 10.1016/j.yexcr.2005.02.015 · DOI
  14. 2005, J. Virol., 79, 12893, 10.1128/JVI.79.20.12893-12904.2005 · DOI
  15. 2005, Proc. Natl. Acad. Sci. USA., 102, 9577, 10.1073/pnas.0502272102 · DOI
  16. 1997, Development., 124, 1561, 10.1242/dev.124.8.1561 · DOI
  17. 2006, J. Immunol., 176, 4894, 10.4049/jimmunol.176.8.4894 · DOI
  18. 2003, J. Biol. Chem., 278, 46541, 10.1074/jbc.M306381200 · DOI
  19. 2006, J. Dent. Res., 85, 49, 10.1177/154405910608500108 · DOI

Cited by

Sorted by citations

Metadata

Authors
Ma, Y., Li, J., Chiu, I., Wang, Y., Sloane, J. A., Lü, J., Kosaras, B., Sidman, R. L., Volpe, J. J., & Vartanian, T.
Affiliations
  • 1Department of Neurology, Beth Israel Deaconess Medical Center, Boston, MA 02115
  • 4Center for Blood Research, Graduate Program in Immunology, Harvard Medical School, Boston, MA 02115
  • 5Pulmonary Center, Boston University School of Medicine, Boston, MA 02118
  • 2Program in Neuroscience
Venue
The Journal of Cell Biology
Publisher
Rockefeller University Press
Published
2006-10-23
Volume
175
Issue
2
Pages
209-215
ISSN
  • 1540-8140
  • 0021-9525
DOI
10.1083/jcb.200606016
PMC
PMC2064562
Cited by
237
References
2
Language
en

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review