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A role for Kalirin-7 in nociceptive sensitization via activity-dependent modulation of spinal synapses.

Lu J, Luo C, Bali KK, Xie RG, Mains RE, Eipper BA, Kuner R - Nat Commun (2015)

Bottom Line: Synaptic plasticity is the cornerstone of processes underlying persistent nociceptive activity-induced changes in normal nociceptive sensitivity.Kalirin-7 is a multifunctional guanine-nucleotide-exchange factor (GEF) for Rho GTPases that is characterized by its localization at excitatory synapses, interactions with glutamate receptors and its ability to dynamically modulate the neuronal cytoskeleton.Here we show that spinally expressed Kalirin-7 is required for persistent nociceptive activity-dependent synaptic long-term potentiation as well as activity-dependent remodelling of synaptic spines in the spinal dorsal horn, thereby orchestrating functional and structural plasticity during the course of inflammatory pain.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Pharmacology, Pharmacology Institute, Medical Faculty Heidelberg, Heidelberg University, Im Neuenheimer Feld 366, 69120 Heidelberg, Germany.

ABSTRACT
Synaptic plasticity is the cornerstone of processes underlying persistent nociceptive activity-induced changes in normal nociceptive sensitivity. Kalirin-7 is a multifunctional guanine-nucleotide-exchange factor (GEF) for Rho GTPases that is characterized by its localization at excitatory synapses, interactions with glutamate receptors and its ability to dynamically modulate the neuronal cytoskeleton. Here we show that spinally expressed Kalirin-7 is required for persistent nociceptive activity-dependent synaptic long-term potentiation as well as activity-dependent remodelling of synaptic spines in the spinal dorsal horn, thereby orchestrating functional and structural plasticity during the course of inflammatory pain.

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Bidirectional modulation of expression of Rac1 in spinal dorsal horn neurons in vivo and its structural and functional impact.(a) Western blotting of spinal lysates for validation of spinal knockdown and overexpression of Rac1 via AAV-mediated shRNA delivery or Rac1 cDNA delivery in comparison with corresponding controls. Lower panel represents quantitative summary of Rac1 expression normalized to corresponding Tubulin control. (b,c) Typical examples of traces derived from Golgi-impregnated spinal neurons (b) and their quantitative summary (c) in mice with spinal AAV-mediated knockdown or overexpression of Rac1, using mice spinally expressing a non-targeting shRNA or EGFP as controls (Scale bar, 50 μm; 15–18 neurons from three animals per treatment group). (d) Attenuation of phase II nocifensive behaviour, but not of phase I, in the formalin test by spinal knockdown of Rac1 (n=7–8 per group). (e) Spinal neuron-specific knockdown of Rac1 led to a reduction of mechanical hypersensitivity induced by intraplantar CFA injection as compared with mice expressing control RNA (n=6 per group). (f) Overexpression of Rac1 in neurons of the spinal dorsal horn led to enhanced basal sensitivity to mechanical nociceptive stimuli (n=6–8 mice). (g) Functional impact of overexpression of Rac1 or EGFP (control) in the spinal dorsal horn of SDH-Kal-7−/− mice on sensitivity to mechanical nociceptive stimuli and CFA-induced hypersensitivity (n=6 mice per group). (h) Quantification of changes in spine density on overexpression of Rac1 or EGFP in spinal neurons lacking Kal-7 at 24 h post-CFA (25–30 neurons were analysed from from animals each per treatment group). *P<0.05 as compared with control group; †P<0.05 as compared with basal condition; two-way ANOVA, Bonferroni post hoc test. Error bars represent s.e.m.
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f4: Bidirectional modulation of expression of Rac1 in spinal dorsal horn neurons in vivo and its structural and functional impact.(a) Western blotting of spinal lysates for validation of spinal knockdown and overexpression of Rac1 via AAV-mediated shRNA delivery or Rac1 cDNA delivery in comparison with corresponding controls. Lower panel represents quantitative summary of Rac1 expression normalized to corresponding Tubulin control. (b,c) Typical examples of traces derived from Golgi-impregnated spinal neurons (b) and their quantitative summary (c) in mice with spinal AAV-mediated knockdown or overexpression of Rac1, using mice spinally expressing a non-targeting shRNA or EGFP as controls (Scale bar, 50 μm; 15–18 neurons from three animals per treatment group). (d) Attenuation of phase II nocifensive behaviour, but not of phase I, in the formalin test by spinal knockdown of Rac1 (n=7–8 per group). (e) Spinal neuron-specific knockdown of Rac1 led to a reduction of mechanical hypersensitivity induced by intraplantar CFA injection as compared with mice expressing control RNA (n=6 per group). (f) Overexpression of Rac1 in neurons of the spinal dorsal horn led to enhanced basal sensitivity to mechanical nociceptive stimuli (n=6–8 mice). (g) Functional impact of overexpression of Rac1 or EGFP (control) in the spinal dorsal horn of SDH-Kal-7−/− mice on sensitivity to mechanical nociceptive stimuli and CFA-induced hypersensitivity (n=6 mice per group). (h) Quantification of changes in spine density on overexpression of Rac1 or EGFP in spinal neurons lacking Kal-7 at 24 h post-CFA (25–30 neurons were analysed from from animals each per treatment group). *P<0.05 as compared with control group; †P<0.05 as compared with basal condition; two-way ANOVA, Bonferroni post hoc test. Error bars represent s.e.m.

Mentions: The structural effects of Kal-7 come about in part via its ability as a GEF to activate RhoG as well as Rac. Whereas RhoG is involved in axonal outgrowth9, Rac1 activation has been functionally linked to dendritic spine plasticity at hippocampal synapses12. Pharmacological blockade of Rac1 has also been reported to affect density of dendritic spines on spinal neurons in neuropathic pain states1920. Here, we observed that knocking down the expression of Rac1 in spinal dorsal horn neurons, using the same in vivo viral strategy described above, diminished nociceptive sensitivity and spine density to the same extent as deletion of Kal-7. Selective knockdown of Rac1 in spinal dorsal horn neurons in vivo was achieved by spinally injecting AAV virions expressing a specific short hairpin RNA (SDH-rAAV-shRNARac1), with AAV virions expressing control RNA being employed as a control (SDH-rAAV-conRNA) (Fig. 4a; Rac1 expression was 65 +/− 9% of control). Neurons expressing Rac1 shRNA demonstrated decreased spine density compared with neurons expressing control RNA (see Fig. 4b for typical examples; Fig. 4c for quantitative summary).


A role for Kalirin-7 in nociceptive sensitization via activity-dependent modulation of spinal synapses.

Lu J, Luo C, Bali KK, Xie RG, Mains RE, Eipper BA, Kuner R - Nat Commun (2015)

Bidirectional modulation of expression of Rac1 in spinal dorsal horn neurons in vivo and its structural and functional impact.(a) Western blotting of spinal lysates for validation of spinal knockdown and overexpression of Rac1 via AAV-mediated shRNA delivery or Rac1 cDNA delivery in comparison with corresponding controls. Lower panel represents quantitative summary of Rac1 expression normalized to corresponding Tubulin control. (b,c) Typical examples of traces derived from Golgi-impregnated spinal neurons (b) and their quantitative summary (c) in mice with spinal AAV-mediated knockdown or overexpression of Rac1, using mice spinally expressing a non-targeting shRNA or EGFP as controls (Scale bar, 50 μm; 15–18 neurons from three animals per treatment group). (d) Attenuation of phase II nocifensive behaviour, but not of phase I, in the formalin test by spinal knockdown of Rac1 (n=7–8 per group). (e) Spinal neuron-specific knockdown of Rac1 led to a reduction of mechanical hypersensitivity induced by intraplantar CFA injection as compared with mice expressing control RNA (n=6 per group). (f) Overexpression of Rac1 in neurons of the spinal dorsal horn led to enhanced basal sensitivity to mechanical nociceptive stimuli (n=6–8 mice). (g) Functional impact of overexpression of Rac1 or EGFP (control) in the spinal dorsal horn of SDH-Kal-7−/− mice on sensitivity to mechanical nociceptive stimuli and CFA-induced hypersensitivity (n=6 mice per group). (h) Quantification of changes in spine density on overexpression of Rac1 or EGFP in spinal neurons lacking Kal-7 at 24 h post-CFA (25–30 neurons were analysed from from animals each per treatment group). *P<0.05 as compared with control group; †P<0.05 as compared with basal condition; two-way ANOVA, Bonferroni post hoc test. Error bars represent s.e.m.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4403379&req=5

f4: Bidirectional modulation of expression of Rac1 in spinal dorsal horn neurons in vivo and its structural and functional impact.(a) Western blotting of spinal lysates for validation of spinal knockdown and overexpression of Rac1 via AAV-mediated shRNA delivery or Rac1 cDNA delivery in comparison with corresponding controls. Lower panel represents quantitative summary of Rac1 expression normalized to corresponding Tubulin control. (b,c) Typical examples of traces derived from Golgi-impregnated spinal neurons (b) and their quantitative summary (c) in mice with spinal AAV-mediated knockdown or overexpression of Rac1, using mice spinally expressing a non-targeting shRNA or EGFP as controls (Scale bar, 50 μm; 15–18 neurons from three animals per treatment group). (d) Attenuation of phase II nocifensive behaviour, but not of phase I, in the formalin test by spinal knockdown of Rac1 (n=7–8 per group). (e) Spinal neuron-specific knockdown of Rac1 led to a reduction of mechanical hypersensitivity induced by intraplantar CFA injection as compared with mice expressing control RNA (n=6 per group). (f) Overexpression of Rac1 in neurons of the spinal dorsal horn led to enhanced basal sensitivity to mechanical nociceptive stimuli (n=6–8 mice). (g) Functional impact of overexpression of Rac1 or EGFP (control) in the spinal dorsal horn of SDH-Kal-7−/− mice on sensitivity to mechanical nociceptive stimuli and CFA-induced hypersensitivity (n=6 mice per group). (h) Quantification of changes in spine density on overexpression of Rac1 or EGFP in spinal neurons lacking Kal-7 at 24 h post-CFA (25–30 neurons were analysed from from animals each per treatment group). *P<0.05 as compared with control group; †P<0.05 as compared with basal condition; two-way ANOVA, Bonferroni post hoc test. Error bars represent s.e.m.
Mentions: The structural effects of Kal-7 come about in part via its ability as a GEF to activate RhoG as well as Rac. Whereas RhoG is involved in axonal outgrowth9, Rac1 activation has been functionally linked to dendritic spine plasticity at hippocampal synapses12. Pharmacological blockade of Rac1 has also been reported to affect density of dendritic spines on spinal neurons in neuropathic pain states1920. Here, we observed that knocking down the expression of Rac1 in spinal dorsal horn neurons, using the same in vivo viral strategy described above, diminished nociceptive sensitivity and spine density to the same extent as deletion of Kal-7. Selective knockdown of Rac1 in spinal dorsal horn neurons in vivo was achieved by spinally injecting AAV virions expressing a specific short hairpin RNA (SDH-rAAV-shRNARac1), with AAV virions expressing control RNA being employed as a control (SDH-rAAV-conRNA) (Fig. 4a; Rac1 expression was 65 +/− 9% of control). Neurons expressing Rac1 shRNA demonstrated decreased spine density compared with neurons expressing control RNA (see Fig. 4b for typical examples; Fig. 4c for quantitative summary).

Bottom Line: Synaptic plasticity is the cornerstone of processes underlying persistent nociceptive activity-induced changes in normal nociceptive sensitivity.Kalirin-7 is a multifunctional guanine-nucleotide-exchange factor (GEF) for Rho GTPases that is characterized by its localization at excitatory synapses, interactions with glutamate receptors and its ability to dynamically modulate the neuronal cytoskeleton.Here we show that spinally expressed Kalirin-7 is required for persistent nociceptive activity-dependent synaptic long-term potentiation as well as activity-dependent remodelling of synaptic spines in the spinal dorsal horn, thereby orchestrating functional and structural plasticity during the course of inflammatory pain.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Pharmacology, Pharmacology Institute, Medical Faculty Heidelberg, Heidelberg University, Im Neuenheimer Feld 366, 69120 Heidelberg, Germany.

ABSTRACT
Synaptic plasticity is the cornerstone of processes underlying persistent nociceptive activity-induced changes in normal nociceptive sensitivity. Kalirin-7 is a multifunctional guanine-nucleotide-exchange factor (GEF) for Rho GTPases that is characterized by its localization at excitatory synapses, interactions with glutamate receptors and its ability to dynamically modulate the neuronal cytoskeleton. Here we show that spinally expressed Kalirin-7 is required for persistent nociceptive activity-dependent synaptic long-term potentiation as well as activity-dependent remodelling of synaptic spines in the spinal dorsal horn, thereby orchestrating functional and structural plasticity during the course of inflammatory pain.

Show MeSH
Related in: MedlinePlus