Tumor necrosis factor receptor 1 is a negative regulator of progenitor proliferation in adult hippocampal neurogenesis

Abstract

Tumor necrosis factor-alpha (TNF-alpha) is a proinflammatory cytokine, acting through the TNF-R1 and TNF-R2 receptors. The two receptors have been proposed to mediate distinct TNF-alpha effects in the CNS, TNF-R1 contributing to neuronal damage and TNF-R2 being neuroprotective. Whether TNF-alpha and its receptors play any role for neurogenesis in the adult brain is unclear. Here we used mouse models with loss of TNF-R1 and TNF-R2 function to establish whether signaling through these receptors could influence hippocampal neurogenesis in vivo under basal conditions, as well as after status epilepticus (SE), which is associated with inflammation and elevated TNF-alpha levels. Notably, in the intact brain, the number of new, mature hippocampal neurons was elevated in TNF-R1(-/-) and TNF-R1/R2(-/-) mice, whereas no significant changes were detected in TNF-R2(-/-) mice. Also after SE, the TNF-R1(-/-) and TNF-R1/R2(-/-) mice produced more new neurons. In contrast, the TNF-R2(-/-) mice showed reduced SE-induced neurogenesis. Cell proliferation in the dentate subgranular zone was elevated in TNF-R1(-/-) and TNF-R1/R2(-/-) mice both under basal conditions and after SE. The TNF-R2(-/-) mice either showed no change or minor decrease of cell proliferation. TNF-R1 and TNF-R2 receptors were expressed by hippocampal progenitors, as assessed with reverse transcription-PCR on sorted or cultured cells and immunocytochemistry on cultures. Our data reveal differential actions of TNF-R1 and TNF-R2 signaling in adult hippocampal neurogenesis and identify for the first time TNF-R1 as a negative regulator of neural progenitor proliferation in both the intact and pathological brain.

Figure 1.

Deletion of TNF-R1 and TNF-R2 differentially affects basal hippocampal neurogenesis. A, Number of new mature neurons (BrdU⁺/NeuN⁺) in the dentate subgranular zone/granule cell layer at 2 weeks after 2 weeks of daily BrdU injections. Means ± SEM; n = 8, 6, and 15 for wild-type, TNF-R1^−/−, and TNF-R2^−/− mice, respectively. *p < 0.05 compared with wild-type, one-way ANOVA with Bonferroni–Dunn post hoc test. B, Confocal images of a BrdU⁺NeuN⁺ cell showing BrdU and NeuN immunoreactivity separately or as merged image. Orthogonal reconstructions from confocal Z-series are presented as viewed in x–z (bottom) and y–z (right) planes.

Figure 2.

Relative distribution of different convulsive behaviors and EEG characteristics for wild-type and TNF-R1^−/−, TNF-R2^−/−, and TNF-R1/R2^−/− mice during the 2 h of self-sustained status epilepticus. Ratings are based on Racine's (1972) scale. Grades 0–2 and 3–5 represent partial and generalized convulsions, respectively. Each segment depicts the mean percentage of time spent exhibiting the behavior. Examples of typical hippocampal EEG recordings during SE are presented for each group.

Figure 3.

Deletion of TNF-R1 and TNF-R2 differentially affects hippocampal neurogenesis after status epilepticus. A, Number of new mature neurons (BrdU⁺/NeuN⁺) in the dentate SGZ/GCL at 4 weeks after 1 week of daily BrdU injections, which started 1 week after 2 h of electrically induced status epilepticus. Means ± SEM; n = 6, 6, and 9 for wild-type, TNF-R1^−/−, and TNF-R2^−/− mice, respectively. *p < 0.05 compared with wild-type, one-way ANOVA with Bonferroni–Dunn post hoc test. B, Photomicrographs showing distribution of BrdU⁺ (red) and NeuN⁺ (green) cells in dentate gyrus. Some BrdU⁺/NeuN⁺ cells (yellow) are depicted by arrows. Scale bar, 70 μm.

Figure 4.

Deletion of both TNF-R1 and TNF-R2 increases hippocampal neurogenesis during basal conditions and after status epilepticus. A, C, Number of new mature neurons (BrdU⁺/NeuN⁺) in the dentate SGZ/GCL of TNF-R1/R2^−/− mice at 2 weeks after 2 weeks of daily BrdU injections (A) and at 4 weeks after 1 week of daily BrdU injections, which started 1 week after 2 h of electrically induced status epilepticus (C). Means ± SEM; n = 5 and 6, and 6 and 10 for wild-type and TNF-R1/R2^−/− mice, respectively. *p < 0.05 compared with wild-type, one-way ANOVA with Bonferroni–Dunn post hoc test. B, Photomicrographs showing distribution of BrdU⁺ (red) and NeuN⁺ (green) cells in dentate gyrus. Some BrdU⁺/NeuN⁺ cells (yellow) are depicted by arrows. Scale bar, 70 μm.

Figure 5.

Deletion of TNF-R1 and TNF-R2 differentially affects hippocampal progenitor proliferation under basal conditions and after status epilepticus. A–F, Number of cells in the dentate SGZ expressing the proliferation markers BrdU (A, D), p-H3 (B, E), and PCNA (C, F) in intact mice (A–C) and at 1 week after electrically induced status epilepticus (D–F). BrdU had been injected four times with 2 h interval, and animals were perfused 2 h thereafter. Means ± SEM; n = 5 and 7, 4 and 7, 5 and 7, and 5 and 7 for wild-type, TNF-R1^−/−, TNF-R2^−/−, and TNF-R1/R2^−/− mice, respectively. *p < 0.05 compared with wild-type, one-way ANOVA with Bonferroni–Dunn post hoc test. G–I, Photomicrographs showing distribution of BrdU⁺ (G), p-H3⁺ (H), and PCNA⁺ cells (I) in SGZ at 1 week after status epilepticus. Some BrdU⁺ (red), p-H3⁺ (black), and PCNA⁺ (black) cells in SGZ are depicted by arrows. Scale bar: G–I, 70 μm.

Figure 6.

Deletion of TNF-R1 and TNF-R2 affects microglia in neurogenic area after status epilepticus. A, B, Numbers of Iba1 immunoreactive cells (showing both active and quiescent microglia) in the dentate SGZ/GCL under basal conditions (A) and at 6 weeks after SE (B). Means ± SEM; n = 11, 6, 10, and 10 for wild-type, TNF-R1^−/−, TNF-R2^−/−, and TNF-R1/R2^−/− mice, respectively. *p < 0.05 compared with wild-type, one-way ANOVA with Bonferroni–Dunn post hoc test. C, Photomicrographs showing distribution of Iba1⁺ cells in dentate hilus, SGZ, and GCL at 6 weeks after SE. Some Iba1⁺ cells (black) are depicted by arrows. Scale bar, 70 μm.

Figure 7.

Hippocampal progenitors express TNF-R1, TNF-R2, and TNF-α. A, Detection of TNF-α, TNF-R1, and TNF-R2 mRNA in sorted hippocampal progenitors from adult nestin–GFP mice and in adult rat hippocampal progenitor cultures (AHP) using RT-PCR. First lane is a DNA ladder to indicate size of amplified products. B–G, TNF-R1 (B) and TNF-R2 (E) immunoreactivity, Hoechst staining (C, F), and merged images (D, G) as observed in cultures of adult rat hippocampal progenitors. Arrows depict one TNF-R1⁺ (B–D) and one TNF-R2⁺ (E–G) cell. Scale bar, 50 μm.