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. 2015 May 5;34(9):1231-43.
doi: 10.15252/embj.201490578. Epub 2015 Mar 12.

FGF22 signaling regulates synapse formation during post-injury remodeling of the spinal cord

Anne Jacobi  1 , Kristina Loy  1 , Anja M Schmalz  1 , Mikael Hellsten  1 , Hisashi Umemori  2 , Martin Kerschensteiner  3 , Florence M Bareyre  4
Affiliations

FGF22 signaling regulates synapse formation during post-injury remodeling of the spinal cord

Anne Jacobi et al. EMBO J. .

Abstract

The remodeling of axonal circuits after injury requires the formation of new synaptic contacts to enable functional recovery. Which molecular signals initiate such axonal and synaptic reorganisation in the adult central nervous system is currently unknown. Here, we identify FGF22 as a key regulator of circuit remodeling in the injured spinal cord. We show that FGF22 is produced by spinal relay neurons, while its main receptors FGFR1 and FGFR2 are expressed by cortical projection neurons. FGF22 deficiency or the targeted deletion of FGFR1 and FGFR2 in the hindlimb motor cortex limits the formation of new synapses between corticospinal collaterals and relay neurons, delays their molecular maturation, and impedes functional recovery in a mouse model of spinal cord injury. These results establish FGF22 as a synaptogenic mediator in the adult nervous system and a crucial regulator of synapse formation and maturation during post-injury remodeling in the spinal cord.

Keywords: axonal remodeling; fibroblast growth factor; functional recovery; spinal cord injury; synapse formation.

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Figures

Figure 1
Figure 1. FGF22 is expressed by interneurons in the healthy and injured adult spinal cord

  1. In situ hybridization of FGF22 mRNA in the spinal cord of FGF22-competent (left panel) and FGF22-deficient (right panel) mice (DH: dorsal horn; VH: ventral horn). Scale bar equals 200 μm (10 μm in inset).

  2. In situ hybridization of a section of the cervical spinal cord at level C4/C5 showing presence of FGF22 mRNA in a long propriospinal neuron (LPSN) retrogradely traced from T12 (LPSN: green; FGF22 mRNA: red; NeuroTrace 435/455: blue). Scale bar equals 20 μm.

  3. Quantification of the percentage of LPSN showing a FGF22 in situ signal in unlesioned mice (‘Ctrl’) and in mice at 3 (‘3w’) and 12 (‘12w’) weeks after spinal cord injury (n > 30 LPSN from 2 to 3 animals per group). Mean ± SEM. No significant differences between the groups were detected (ANOVA followed by Tukey tests).

  4. Images of LPSN (red) before (left panel) and after (right panel) laser microdissection of a single neuron (arrow, asterisk and dotted line in right panel indicate previous location of the microdissected neuron). Scale bar equals 40 μm.

  5. Quantification of the single-cell PCR analysis of FGF22 mRNA expression in LPSN dissected from unlesioned mice (‘Ctrl’) and from mice at 3 (‘3w’) and 12 (‘12w’) weeks after spinal cord injury (n = 7–8 LPSN per group). Mean ± SEM. No significant differences between the groups were detected (ANOVA followed by Tukey tests).

Figure 2
Figure 2. FGF22 deficiency impairs bouton formation and circuit remodeling after spinal cord injury

  1. Schematic representation of CST detour circuit formation following a mid-thoracic dorsal bilateral hemisection of the spinal cord.

  2. Confocal images of hindlimb CST collaterals exiting the main CST tract (arrows) in the cervical spinal cord 3 weeks following a T8 dorsal bilateral hemisection in FGF22-competent (left panel) and FGF22-deficient (right panel) mice. Scale bar equals 40 μm.

  3. Quantification of the number of exiting hindlimb CST collaterals per labeled hindlimb CST fiber at 3 weeks following T8 dorsal bilateral hemisection in FGF22-competent and FGF22-deficient mice (n = 8 animals per group). Mean ± SEM. No significant differences between the groups were detected (unpaired two-tailed t-test).

  4. Confocal images showing putative synaptic boutons (arrows) on newly formed cervical hindlimb CST collaterals at 3 weeks following spinal cord injury in FGF22-competent (left panel) and FGF22-deficient (right panel) mice. Scale bar equals 20 μm.

  5. Quantification of bouton density on newly formed cervical hindlimb CST collaterals in FGF22-competent and FGF22-deficient mice (n = 8 animals per group) at 3 weeks after injury. Mean ± SEM. *P = 0.0244 (unpaired two-tailed t-test).

  6. 3D Rendering of a confocal image stack that illustrates putative synaptic contacts between CST collaterals (green) and LPSN (red) counterstained with NeuroTrace 435/455 (blue). Scale bar equals 30 μm.

  7. Quantification of the percentage of LPSN contacted by cervical hindlimb CST collaterals in FGF22-competent and FGF22-deficient mice at 3 weeks after injury (n = 8 animals per group). Mean ± SEM. ***P = 0.0001 (unpaired two-tailed t-test).

Figure 3
Figure 3. FGFR1 and FGFR2 are expressed in adult cortical projection neurons

  1. In situ hybridization of FGFR1 (top) and FGFR2 (bottom) mRNA in FGFR-competent mice. Scale bar equals 200 μm.

  2. In situ hybridization of FGFR1 (top) and FGFR2 (bottom) mRNA in forebrain FGFR1 (top)- and FGFR2 (bottom)-deficient mice. Scale bar equals 200 μm.

  3. Retrograde labeling of CST projection neurons with dextran conjugated with Texas Red® (green) shows that many of these neurons express FGFR1 (red, top) and FGFR2 mRNA (red, bottom; insets in top and bottom panels are twofold magnification of boxed areas). Scale bar equals 100 μm (20 μm in insets).

  4. Quantification of the percentage of CST projection neurons in layer V of the cortex expressing FGFR1 (white bar) and FGFR2 mRNA (gray bar; n = 5 animals per group). Mean ± SEM.

  5. Quantification of the intensity of the in situ signal for FGFR1 mRNA in FGFR2-deficient mice (green bar) and FGFR2 mRNA in FGFR1-deficient mice (blue bar) normalized to the signal intensity measured in the respective FGFR-competent control group (white bars; n = 3 animals per group). Mean ± SEM. No significant differences between the groups were detected (unpaired two-tailed t-tests).

Figure 4
Figure 4. Deletion of forebrain FGFR1 and FGFR2 expression impairs bouton formation and circuit remodeling after spinal cord injury

  1. Confocal images of hindlimb CST collaterals exiting the main CST tract (arrows) in the cervical spinal cord 3 weeks following T8 dorsal bilateral hemisection in FGFR-competent (left panel), forebrain FGFR1-deficient (middle panel), and forebrain FGFR2-deficient (right panel) mice. Scale bar equals 40 μm.

  2. Confocal images of hindlimb CST collaterals exiting the main CST tract (arrows) in the cervical spinal cord 3 weeks following T8 dorsal bilateral hemisection in FGFR-competent (left panel) and hindlimb motor cortex FGFR1/FGFR2 double-deficient (right panel) mice. Scale bar equals 40 μm.

  3. Quantification of the number of exiting hindlimb CST collaterals at 3 weeks following T8 dorsal bilateral hemisection in forebrain FGFR single-deficient mice, hindlimb motor cortex FGFR1/FGFR2 double-deficient mice, and the corresponding FGFR-competent control mice (n = 6–15 animals per group). Mean ± SEM. *P < 0.05; ***P < 0.001 (ANOVA followed by Tukey tests for FGFR-competent versus FGFR single-deficient mice). No significant differences were found between FGFR-competent and FGFR1/FGFR2 double-deficient mice (unpaired two-tailed t-tests).

  4. Confocal images showing putative synaptic boutons (arrows) on newly formed cervical hindlimb CST collaterals at 3 weeks following spinal cord injury in FGFR-competent (left panel), forebrain FGFR1-deficient (second panel from left), forebrain FGFR2-deficient (second panel from right), and hindlimb motor cortex FGFR1/FGFR2 double-deficient (right panel) mice. Scale bar equals 20 μm.

  5. Quantification of the bouton density on newly formed cervical hindlimb CST collaterals in FGFR-competent, forebrain single FGFR-deficient, and hindlimb motor cortex FGFR1/FGFR2 double-deficient mice (n = 6–16 animals per group). Mean ± SEM. *P < 0.05, **P < 0.01 (ANOVA followed by Tukey tests in case of multiple group comparisons, e.g. FGFR-competent versus FGFR single-deficient mice). **P = 0.0028 (unpaired two-tailed t-tests for comparisons of FGFR-competent versus FGFR1/FGFR2 double-deficient mice).

  6. Quantification of the percentage of LPSN contacted by hindlimb CST collaterals in FGFR-competent, forebrain single FGFR-deficient, and hindlimb motor cortex FGFR1/FGFR2 double-deficient mice (n = 6–16 animals per group). ***P < 0.0001 (unpaired two-tailed t-tests for comparisons of controls versus FGFR1/FGFR2 double-deficient mice). No significant differences were found between FGFR-competent and FGFR single-deficient mice (ANOVA followed by Tukey tests).

Figure 5
Figure 5. Deletion of FGF22 or its receptors delays synapse maturation following spinal cord injury

  1. Confocal image of synaptic contacts (arrows) between a CST collateral (green) and a LPSN (blue) that show bassoon immunoreactivity (red). Right images are magnification (two and a half-fold) of the area boxed on the left. Scale bar equals 25 μm.

  2. Quantification of the percentage of boutons on cervical hindlimb CST collaterals that are immunoreactive for bassoon at 3 weeks (left) and 12 weeks (right) after spinal cord injury in FGF22-deficient, forebrain FGFR1-deficient, forebrain FGFR2-deficient and hindlimb motor cortex FGFR1/FGFR2 double-deficient mice compared to the respective FGF22- and FGFR-competent control mice. A minimum of 100 boutons per mouse were evaluated for 3 mice per group. ***P < 0.0001 FGFR-competent versus FGFR1/FGFR2 double-deficient mice at 3 weeks (unpaired two-tailed t-tests), **P = 0.002 FGF-competent versus FGF22 deficient at 3 weeks (unpaired two-tailed t-tests), *P < 0.05 FGFR-competent versus FGFR2 single-deficient mice at 12 weeks (one-way ANOVA followed by Tukey tests), ***P = 0.001 FGFR-competent versus FGFR1/FGFR2 double-deficient mice at 12 weeks (unpaired two-tailed t-tests).

  3. Confocal image of synaptic contacts (arrows) between a CST collateral (green) and a LPSN (blue) that show synapsin I immunoreactivity (red). Right images are magnification (two and a half-fold) of the area boxed on the left. Scale bar equals 25 μm.

  4. Quantification of the percentage of boutons on cervical hindlimb CST collaterals that are immunoreactive for synapsin I at 3 weeks (left) and 12 weeks (right) after spinal cord injury in FGF22-deficient, forebrain FGFR1-deficient, forebrain FGFR2-deficient and hindlimb motor cortex FGFR1/FGFR2 double-deficient mice compared to the respective FGF22 and FGFR-competent control mice. A minimum of 100 boutons per mouse were evaluated for 3 mice per group. ***P < 0.001 FGFR-competent versus FGFR1 and FGFR2 single-deficient mice at 3 weeks (one-way ANOVA followed by Tukey tests). ***P = 0.0001 FGFR-competent mice versus FGFR1/FGFR2 double-deficient mice at 3 weeks (unpaired two-tailed t-tests), P = 0.00004 FGF-competent mice versus FGF22-deficient mice at 3 weeks (unpaired two-tailed t-tests). ***P < 0.001 FGFR-competent versus FGFR2 single-deficient mice (one-way ANOVA followed by Tukey tests). ***P = 0.0006 FGFR-competent mice versus FGFR1/FGFR2 double-deficient mice at 12 weeks (unpaired two-tailed t-tests), *P = 0.027 FGF-competent mice versus FGF22-deficient mice at 12 weeks (unpaired two-tailed t-tests).

Figure 6
Figure 6. Genetic disruption of FGF22 signaling impedes functional recovery following spinal cord injury

  1. Image of a spinal cord injured mouse performing the irregular ladder rung test that assesses recovery of CST function. Scale bar equals 1 cm.

  2. Quantification of the functional recovery in the ladder rung test (regular walk, left panels; irregular walk, right panels) in FGF22-deficient (top panels, red bars), and hindlimb motor cortex FGFR1/FGFR2 double-deficient (bottom panels, blue bars) mice and the respective FGF22- and FGFR-competent control mice (white bars) before (‘Pre’) and 2 (‘2 wks’) and 3 (‘3 wks’) weeks after a spinal cord injury (n = 7–10 animals per group). *P < 0.05, **P < 0.01 (repeated-measure ANOVA followed by Bonferroni tests).

  3. Image of a spinal cord injured mouse walking on the catwalk that assesses locomotor recovery. Illumination of the paws from below allows to determine the paw angle body axis by relating the axis of the paw (line shown magnified in the right panel) to the axis of the body (line shown in left panel).

  4. Quantification of the paw angle body axis of the hindpaws in FGF22-deficient (top panel, red bars) and hindlimb motor cortex FGFR1/FGFR2 double-deficient (bottom panel, blue bars) mice and respective FGF22- and FGFR-competent control mice (white bars) before (‘Pre’) and 2 (‘2 wks’) and 3 (‘3 wks’) weeks after a spinal cord injury. Between 13 and 40 steps were analyzed per group and timepoint (n = 10–15 animals per group). **P < 0.01, ***P < 0.001 (two-way ANOVA followed by Bonferroni tests).

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