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. 2002 Feb 1;22(3):854-62.
doi: 10.1523/JNEUROSCI.22-03-00854.2002.

Tumor necrosis factor inhibits neurite outgrowth and branching of hippocampal neurons by a rho-dependent mechanism

Harald Neumann  1 , Rudiger Schweigreiter, Toshihide Yamashita, Katja Rosenkranz, Hartmut Wekerle, Yves-Alain Barde
Affiliations

Tumor necrosis factor inhibits neurite outgrowth and branching of hippocampal neurons by a rho-dependent mechanism

Harald Neumann et al. J Neurosci. .

Abstract

In response to injury and inflammation of the CNS, brain cells including microglia and astrocytes secrete tumor necrosis factor-alpha (TNF). This pro-inflammatory cytokine has been implicated in both neuronal cell death and survival. We now provide evidence that TNF affects the formation of neurites. Neurons cultured on astrocytic glial cells exhibited reduced outgrowth and branching of neurites after addition of recombinant TNF or prestimulation of glial cells to secrete TNF. This effect was absent in neurons of TNF receptor-deficient mice cultured on prestimulated glia of wild-type mice and was reverted by blocking TNF with soluble TNF receptor IgG fusion protein. TNF activated in neurons the small GTPase RhoA. By inactivating Rho with C3 transferase, the inhibitory effect of TNF on neurite outgrowth and branching was abolished. These results suggest that glia-derived TNF, as part of an injury or inflammatory process, can inhibit neurite elongation and branching during development and regeneration.

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Figures

Fig. 1.
Fig. 1.
TNF receptor gene transcription in hippocampal neurons. Single-cell RT-PCR of cultured primary hippocampal neurons. Gene transcripts for TNFRI and TNFRII were detected in the majority of neurons. Co-amplification of gene transcripts for GAPDH served as a control. M, Molecular weight marker ΦX174/HaeIII; C, negative PCR control;N1–N9, samples of single neurons.
Fig. 2.
Fig. 2.
Immunodetection of TNFRI and TNFRII molecules in primary hippocampal neurons. Neurons were immunolabeled with rat monoclonal antibodies directed against TNFRI or TNFRII and rat control antibodies. Neurons were subsequently double labeled with antibody directed against β-tubulin III. Scale bar, 10 μm.
Fig. 3.
Fig. 3.
Computer-assisted tracing of β-tubulin III-labeled neurites. Primary hippocampal neurons were cultured for 16 hr on astrocyte-enriched glial cells. Neurons were immunolabeled with antibodies against β-tubulin III, and neurites of captured images were traced with image analysis software. Treatment with 10 ng/ml TNF reduced neurite outgrowth and branching of neurites. Scale bar, 10 μm.
Fig. 4.
Fig. 4.
Morphometric analyses of primary hippocampal neurons cultured for 16 hr on astrocyte-enriched glia. Neurons and glial cells were derived from E16 C57BL/6 mice (Wild-type) or C57BL/6 TNFRI- plus TNFRII-deficient mice (Neurons TNFR −/−, Glia TNFR −/−). The cultures were either untreated (gray columns) or treated for 16 hr with 10 ng/ml TNF (black columns). TNF reduced the total neurite length, axonal length, and number of branch points of neurons derived from C57BL/6 mice, but not TNFR −/− mice. The reduction in neurite outgrowth and branching was independent of the TNF receptor expression of the glia. Data are presented as mean ± SEM of three independent experiments.
Fig. 5.
Fig. 5.
Detection of TNF gene transcripts, membrane-bound TNF, and TNF released in response to cytokine stimulation of glia. Astrocyte-enriched glial cells were untreated (Con) or treated with 100 U/ml IFN-γ (IFN), 10 ng/ml IL-1β (IL1), or IFN-γ plus IL-1β (IFN/IL1) for 24 hr. A, Analysis of TNF gene transcripts by RT-PCR. Glia showed low amounts of TNF gene transcripts after treatment with IFN-γ or IL-1β alone, whereas high amounts of TNF gene transcripts were detected after combined treatment with IFN-γ and ILl-1β. Coamplification of gene transcripts for GAPDH served as a control. M, Molecular weight marker ΦX174/HaeIII; Neg, negative PCR control. B, Membrane-bound TNF was detected on astrocyte-enriched glial cells with fluorescence-labeled rabbit antibody directed against TNF and flow cytometry. Data are presented as mean ± SEM of three independent experiments. C, Soluble TNF released within 6 hr was detected in the supernatant of cytokine-stimulated glia. Data are presented as mean ± SEM of three independent experiments.
Fig. 6.
Fig. 6.
Morphometric analyses of primary hippocampal neurons cultured for 16 hr on reactive glia that has been pretreated for 24 hr with IFN-γ and IL-1β to induce TNF production. Neurons were derived from E16 C57BL/6 mice (Neurons wild-type) or C57BL/6 TNFRI- plus TNFRII-deficient mice (Neurons TNFR −/−). The cultures were treated for 16 hr with control IgG (gray columns) or with TNF receptor IgG fusion protein (TNFR-IgG, black columns). Blockade of TNF with TNFR-IgG antagonized the reduced total neurite length, axonal length, and number of branch points of neurons derived from wild-type, but not TNFR −/− mice. Data are presented as mean ± SEM of three independent experiments.
Fig. 7.
Fig. 7.
Morphometric analyses of primary hippocampal neurons cultured for 16 hr on unstimulated glia. Neurons were derived from E16 C57BL/6 mice (Neurons wild-type) or C57BL/6 TNFRI- plus TNFRII-deficient mice (Neurons TNFR −/−). The cultures were treated for 16 hr with control IgG (gray columns) or with TNF receptor IgG fusion protein (TNFR-IgG, black columns). Blockade of TNF with TNFR-IgG did not change the total neurite length, axonal length, and number of branch points of neurons cultured on unstimulated glial cells. Data are presented as mean ± SEM of three independent experiments.
Fig. 8.
Fig. 8.
RhoA activation in primary hippocampal neurons after TNF treatment. Neurons were treated with 10 ng/ml TNF for different time periods (15 min, and 1, 4, and 16 hr), and GTP–RhoA was detected by Western blotting after precipitation with Rhotekin. TNF activated RhoA within 1 hr, showing increasing levels up to 16 hr. Activation of RhoA with 10% serum (10 min) served as a positive control.
Fig. 9.
Fig. 9.
Morphometric analyses of primary hippocampal neurons cultured for 16 hr on astrocytes. Neurons were derived from E16 C57BL/6 mice and either triturated together with C3 transferase (Neurons C3 treated) or with buffer alone (Neurons buffer). The cultures were untreated (gray columns) or treated for 16 hr with 10 ng/ml TNF (black columns). TNF-mediated reduction in total neurite length, axonal length, and number of branch points was antagonized in C3-treated but not control buffer-treated neurons. Data are presented as mean ± SEM of three independent experiments.
Fig. 10.
Fig. 10.
Morphometric analyses of primary hippocampal neurons cultured for 16 hr on glia that has been pretreated for 24 hr with IFN-γ and IL-1β to induce TNF production. Neurons were derived from E16 C57BL/6 mice and either triturated together with C3 transferase (Neurons C3 treated) or with buffer alone (Neurons buffer). The cultures were treated for 16 hr with control IgG (gray columns) or with TNF receptor IgG fusion protein (TNFR-IgG, black columns). Trituration of neurons together with C3 transferase antagonized the reduced total neurite length, axonal length, and number of branch points. Data are presented as mean ± SEM of three independent experiments.
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