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Conditional gene ablation of Stat3 reveals differential signaling requirements for survival of motoneurons during development and after nerve injury in the adult.

Schweizer U, Gunnersen J, Karch C, Wiese S, Holtmann B, Takeda K, Akira S, Sendtner M - J. Cell Biol. (2002)

Bottom Line: In contrast, motoneuron survival is significantly reduced after facial nerve lesion in the adult.Stat3 is essential for upregulation of Reg-2 and Bcl-xl expression in lesioned motoneurons.Our data show that Stat3 activation plays an essential role for motoneuron survival after nerve lesion in postnatal life but not during embryonic development, indicating that signaling requirements for motoneuron survival change during maturation.

View Article: PubMed Central - PubMed

Affiliation: Institute for Clinical Neurobiology, University of Würzburg, 97080 Würzburg, Germany.

ABSTRACT
Members of the ciliary neurotrophic factor (CNTF)/leukemia inhibitory factor (LIF)/cardiotrophin gene family are potent survival factors for embryonic and lesioned motoneurons. These factors act via receptor complexes involving gp130 and LIFR-beta and ligand binding leads to activation of various signaling pathways, including phosphorylation of Stat3. The role of Stat3 in neuronal survival was investigated in mice by Cre-mediated gene ablation in motoneurons. Cre is expressed under the neurofilament light chain (NF-L) promoter, starting around E12 when these neurons become dependent on neurotrophic support. Loss of motoneurons during the embryonic period of naturally occurring cell death is not enhanced in NF-L-Cre; Stat3(flox/KO) mice although motoneurons isolated from these mice need higher concentrations of CNTF for maximal survival in culture. In contrast, motoneuron survival is significantly reduced after facial nerve lesion in the adult. These neurons, however, can be rescued by the addition of neurotrophic factors, including CNTF. Stat3 is essential for upregulation of Reg-2 and Bcl-xl expression in lesioned motoneurons. Our data show that Stat3 activation plays an essential role for motoneuron survival after nerve lesion in postnatal life but not during embryonic development, indicating that signaling requirements for motoneuron survival change during maturation.

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Reg-2 expression is induced in axotomized facial motoneurons in a Stat3-dependent manner. (A) RT-PCR analysis using total RNA derived from acutely isolated facial nuclei revealed an increase of Reg-2 mRNA 2 d after facial nerve transection. In NF-L–Cre; Stat3flox/KO mice, Reg-2 mRNA levels are only 34% of the amount in the NF-L–Cre; Stat3flox/wt littermates. This indicates that Stat3 is involved in the upregulation of Reg-2 in motoneurons after axotomy (n = 5, P < 0.05, ANOVA with Bonferroni's post hoc comparison). Results shown are from one representative experiment of three experiments with similar results. (B) In situ hybridization reveals that Reg-2 is specifically expressed in facial motoneurons, but not in surrounding glial cells. The bottom panel shows an adjacent section hybridized with the sense probe. Bar, 100 μm.
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fig5: Reg-2 expression is induced in axotomized facial motoneurons in a Stat3-dependent manner. (A) RT-PCR analysis using total RNA derived from acutely isolated facial nuclei revealed an increase of Reg-2 mRNA 2 d after facial nerve transection. In NF-L–Cre; Stat3flox/KO mice, Reg-2 mRNA levels are only 34% of the amount in the NF-L–Cre; Stat3flox/wt littermates. This indicates that Stat3 is involved in the upregulation of Reg-2 in motoneurons after axotomy (n = 5, P < 0.05, ANOVA with Bonferroni's post hoc comparison). Results shown are from one representative experiment of three experiments with similar results. (B) In situ hybridization reveals that Reg-2 is specifically expressed in facial motoneurons, but not in surrounding glial cells. The bottom panel shows an adjacent section hybridized with the sense probe. Bar, 100 μm.

Mentions: It has been shown previously that Reg-2, a Schwann cell mitogen (Livesey et al., 1997) and motoneuron survival factor (Nishimune et al., 2000), is expressed in spinal motoneurons upon CNTF addition via LIFR-β–mediated signal transduction. Reg-2 acts on isolated motoneurons in an autocrine and paracrine mode to enhance survival (Nishimune et al., 2000). The Reg-2 promoter contains three Stat3 binding sites and is up-regulated in response to Stat3 activation (Dusetti et al., 1995). Therefore, Reg-2 produced by Stat3 bearing motoneurons could enhance the survival of Stat3 motoneurons (that cannot make Reg-2 on their own) in a paracrine manner. Stat3 phosphorylation in facial motoneurons peaks at 2 d after axotomy (Schwaiger et al., 2000). Therefore, we have chosen this time point to measure expression of Reg-2 in the facial nucleus by semiquantitative RT-PCR. After facial nerve lesion in NF-L–Cre; Stat3flox/wt mice, Reg-2 mRNA is induced 11.1 ± 4.67-fold in the facial nucleus on the lesioned side, demonstrating that Reg-2 expression is induced by axotomy in facial motoneurons as shown previously for spinal motoneurons (Livesey et al., 1997). In NF-L–Cre; Stat3flox/KO mice Reg-2 expression increases 3.82 ± 2.6-fold compared with the unlesioned control (means calculated from three experiments). Reg-2 mRNA levels in NF-L–Cre; Stat3flox/KO mice are only 34% of those observed in NF-L–Cre; Stat3flox/wt mice (n = 4–5, P < 0.05; Fig. 5 A). Therefore, Stat3 is a likely regulator of Reg-2 expression in vivo. In situ hybridization on sections including both facial nuclei on the lesioned and the control side showed that Reg-2 expression was confined to motoneurons (Fig. 5 B).


Conditional gene ablation of Stat3 reveals differential signaling requirements for survival of motoneurons during development and after nerve injury in the adult.

Schweizer U, Gunnersen J, Karch C, Wiese S, Holtmann B, Takeda K, Akira S, Sendtner M - J. Cell Biol. (2002)

Reg-2 expression is induced in axotomized facial motoneurons in a Stat3-dependent manner. (A) RT-PCR analysis using total RNA derived from acutely isolated facial nuclei revealed an increase of Reg-2 mRNA 2 d after facial nerve transection. In NF-L–Cre; Stat3flox/KO mice, Reg-2 mRNA levels are only 34% of the amount in the NF-L–Cre; Stat3flox/wt littermates. This indicates that Stat3 is involved in the upregulation of Reg-2 in motoneurons after axotomy (n = 5, P < 0.05, ANOVA with Bonferroni's post hoc comparison). Results shown are from one representative experiment of three experiments with similar results. (B) In situ hybridization reveals that Reg-2 is specifically expressed in facial motoneurons, but not in surrounding glial cells. The bottom panel shows an adjacent section hybridized with the sense probe. Bar, 100 μm.
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Related In: Results  -  Collection

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fig5: Reg-2 expression is induced in axotomized facial motoneurons in a Stat3-dependent manner. (A) RT-PCR analysis using total RNA derived from acutely isolated facial nuclei revealed an increase of Reg-2 mRNA 2 d after facial nerve transection. In NF-L–Cre; Stat3flox/KO mice, Reg-2 mRNA levels are only 34% of the amount in the NF-L–Cre; Stat3flox/wt littermates. This indicates that Stat3 is involved in the upregulation of Reg-2 in motoneurons after axotomy (n = 5, P < 0.05, ANOVA with Bonferroni's post hoc comparison). Results shown are from one representative experiment of three experiments with similar results. (B) In situ hybridization reveals that Reg-2 is specifically expressed in facial motoneurons, but not in surrounding glial cells. The bottom panel shows an adjacent section hybridized with the sense probe. Bar, 100 μm.
Mentions: It has been shown previously that Reg-2, a Schwann cell mitogen (Livesey et al., 1997) and motoneuron survival factor (Nishimune et al., 2000), is expressed in spinal motoneurons upon CNTF addition via LIFR-β–mediated signal transduction. Reg-2 acts on isolated motoneurons in an autocrine and paracrine mode to enhance survival (Nishimune et al., 2000). The Reg-2 promoter contains three Stat3 binding sites and is up-regulated in response to Stat3 activation (Dusetti et al., 1995). Therefore, Reg-2 produced by Stat3 bearing motoneurons could enhance the survival of Stat3 motoneurons (that cannot make Reg-2 on their own) in a paracrine manner. Stat3 phosphorylation in facial motoneurons peaks at 2 d after axotomy (Schwaiger et al., 2000). Therefore, we have chosen this time point to measure expression of Reg-2 in the facial nucleus by semiquantitative RT-PCR. After facial nerve lesion in NF-L–Cre; Stat3flox/wt mice, Reg-2 mRNA is induced 11.1 ± 4.67-fold in the facial nucleus on the lesioned side, demonstrating that Reg-2 expression is induced by axotomy in facial motoneurons as shown previously for spinal motoneurons (Livesey et al., 1997). In NF-L–Cre; Stat3flox/KO mice Reg-2 expression increases 3.82 ± 2.6-fold compared with the unlesioned control (means calculated from three experiments). Reg-2 mRNA levels in NF-L–Cre; Stat3flox/KO mice are only 34% of those observed in NF-L–Cre; Stat3flox/wt mice (n = 4–5, P < 0.05; Fig. 5 A). Therefore, Stat3 is a likely regulator of Reg-2 expression in vivo. In situ hybridization on sections including both facial nuclei on the lesioned and the control side showed that Reg-2 expression was confined to motoneurons (Fig. 5 B).

Bottom Line: In contrast, motoneuron survival is significantly reduced after facial nerve lesion in the adult.Stat3 is essential for upregulation of Reg-2 and Bcl-xl expression in lesioned motoneurons.Our data show that Stat3 activation plays an essential role for motoneuron survival after nerve lesion in postnatal life but not during embryonic development, indicating that signaling requirements for motoneuron survival change during maturation.

View Article: PubMed Central - PubMed

Affiliation: Institute for Clinical Neurobiology, University of Würzburg, 97080 Würzburg, Germany.

ABSTRACT
Members of the ciliary neurotrophic factor (CNTF)/leukemia inhibitory factor (LIF)/cardiotrophin gene family are potent survival factors for embryonic and lesioned motoneurons. These factors act via receptor complexes involving gp130 and LIFR-beta and ligand binding leads to activation of various signaling pathways, including phosphorylation of Stat3. The role of Stat3 in neuronal survival was investigated in mice by Cre-mediated gene ablation in motoneurons. Cre is expressed under the neurofilament light chain (NF-L) promoter, starting around E12 when these neurons become dependent on neurotrophic support. Loss of motoneurons during the embryonic period of naturally occurring cell death is not enhanced in NF-L-Cre; Stat3(flox/KO) mice although motoneurons isolated from these mice need higher concentrations of CNTF for maximal survival in culture. In contrast, motoneuron survival is significantly reduced after facial nerve lesion in the adult. These neurons, however, can be rescued by the addition of neurotrophic factors, including CNTF. Stat3 is essential for upregulation of Reg-2 and Bcl-xl expression in lesioned motoneurons. Our data show that Stat3 activation plays an essential role for motoneuron survival after nerve lesion in postnatal life but not during embryonic development, indicating that signaling requirements for motoneuron survival change during maturation.

Show MeSH
Related in: MedlinePlus