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Defective Gpsm2/G α i3 signalling disrupts stereocilia development and growth cone actin dynamics in Chudley-McCullough syndrome

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ABSTRACT

Mutations in GPSM2 cause Chudley-McCullough syndrome (CMCS), an autosomal recessive neurological disorder characterized by early-onset sensorineural deafness and brain anomalies. Here, we show that mutation of the mouse orthologue of GPSM2 affects actin-rich stereocilia elongation in auditory and vestibular hair cells, causing deafness and balance defects. The G-protein subunit Gαi3, a well-documented partner of Gpsm2, participates in the elongation process, and its absence also causes hearing deficits. We show that Gpsm2 defines an ∼200 nm nanodomain at the tips of stereocilia and this localization requires the presence of Gαi3, myosin 15 and whirlin. Using single-molecule tracking, we report that loss of Gpsm2 leads to decreased outgrowth and a disruption of actin dynamics in neuronal growth cones. Our results elucidate the aetiology of CMCS and highlight a new molecular role for Gpsm2/Gαi3 in the regulation of actin dynamics in epithelial and neuronal tissues.

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Gpsm2 mutation leads to hypoplasia of the corpus callosum and affects growth cone outgrowth.(a) Hematoxylin staining of coronal sections from P6 Gpsm2Emx1 control (upper panel) and cKO brains (lower panel) at a caudal levels. Note the abscence of corpus callossum (CC) in the cKO mouse (inset). Scale bar, 1 mm, n=4 independent experiments. (b,c) In cultured hippocampal neurons at DIV2, N-cadherin-dependent outgrowth was reduced in Gpsm2 cKOs, but not in Gnai3 cKOs (b). The reduction in outgrowth was maintained on a laminin substrate in Gpsm2 cKOs (c). Data from three to six independent experiments are presented as whisker box plots (min/max) (n=number of growth cones). ***P<0.001 with unpaired Student's t-test. (d) Images from three time points (0, 15 and 30 min) of a time-lapse movie from 2 DIV control and Gpsm2 cKO hippocampal neurons plated on N-cadherin-coated glass and showing the difference in distance covered (dotted lines and double-headed arrows). Scale bar=10 μm. (e) Quantification of the average number of pauses (±s.e.m.) during a 30 min period of growth cones from control (n=31 neurons) compared with Gpsm2 cKO (n=51 neurons), from four independent experiments. The Gpsm2 cKO growth cones pause more than controls. ***P<0.001, *P<0.05 with unpaired Student's t-test. (f) Cumulated distance covered by control and Gpsm2 cKO growth cones over a 30 min period. Some pauses are indicated with blue arrows.
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f7: Gpsm2 mutation leads to hypoplasia of the corpus callosum and affects growth cone outgrowth.(a) Hematoxylin staining of coronal sections from P6 Gpsm2Emx1 control (upper panel) and cKO brains (lower panel) at a caudal levels. Note the abscence of corpus callossum (CC) in the cKO mouse (inset). Scale bar, 1 mm, n=4 independent experiments. (b,c) In cultured hippocampal neurons at DIV2, N-cadherin-dependent outgrowth was reduced in Gpsm2 cKOs, but not in Gnai3 cKOs (b). The reduction in outgrowth was maintained on a laminin substrate in Gpsm2 cKOs (c). Data from three to six independent experiments are presented as whisker box plots (min/max) (n=number of growth cones). ***P<0.001 with unpaired Student's t-test. (d) Images from three time points (0, 15 and 30 min) of a time-lapse movie from 2 DIV control and Gpsm2 cKO hippocampal neurons plated on N-cadherin-coated glass and showing the difference in distance covered (dotted lines and double-headed arrows). Scale bar=10 μm. (e) Quantification of the average number of pauses (±s.e.m.) during a 30 min period of growth cones from control (n=31 neurons) compared with Gpsm2 cKO (n=51 neurons), from four independent experiments. The Gpsm2 cKO growth cones pause more than controls. ***P<0.001, *P<0.05 with unpaired Student's t-test. (f) Cumulated distance covered by control and Gpsm2 cKO growth cones over a 30 min period. Some pauses are indicated with blue arrows.

Mentions: In addition to early-onset sensorineural deafness, patients with CMCS also display specific brain malformations on magnetic resonance images; hypoplasia of the CC being a hallmark of this pathology1. To evaluate the impact of Gpsm2 on CC development, we generated Emx1-Cre*Gpsm2 cKOs (Gpsm2Emx1), deleting Gpsm2 in the early and dorsal telencephalon. Analysis of brains from these cKOs confirmed the existence of a caudal CC agenesis (Fig. 7a), as reported in CMCS patients. CC defects can result from a disruption in neuronal progenitors or defects in axonal elongation and guidance, which are dependent upon the microtubule and actin cytoskeleton34. Neurite elongation is due to the motility of the growth cone driven in part by protrusive forces generated by actin polymerization at the leading edge3435 and the existence of a retrograde flow of actin resulting from a balance of filament polymerization and depolymerization, among other factors3637. We therefore hypothesized that actin dynamics could be impaired in mutant Gpsm2 neurons and affect outgrowth. Measures of growth cone locomotion on N-cadherin-coated substrates showed that outgrowth of Gpsm2 cKOs neurons was reduced by 37% compared with controls (control 0.97±0.04 μm min−1 versus cKO 0.61±0.02 μm min−1), whereas the speed of growth cones from Gnai3 cKOs was indistinguishable from controls (control 0.92±0.02 μm min−1; cKO 0.89±0.02 μm min−1) (Fig. 7b). This Gpsm2 cKO phenotype was also observed on a laminin-coated substrate, suggesting that the outgrowth reduction was not specific to an N-cadherin substrate (Fig. 7c). The number of pauses the growth cone made during the 30 min time-lapses on N-cadherin substrate was increased in Gpsm2 cKO neurons (Fig. 7d–f, Supplementary Movie 1).


Defective Gpsm2/G α i3 signalling disrupts stereocilia development and growth cone actin dynamics in Chudley-McCullough syndrome
Gpsm2 mutation leads to hypoplasia of the corpus callosum and affects growth cone outgrowth.(a) Hematoxylin staining of coronal sections from P6 Gpsm2Emx1 control (upper panel) and cKO brains (lower panel) at a caudal levels. Note the abscence of corpus callossum (CC) in the cKO mouse (inset). Scale bar, 1 mm, n=4 independent experiments. (b,c) In cultured hippocampal neurons at DIV2, N-cadherin-dependent outgrowth was reduced in Gpsm2 cKOs, but not in Gnai3 cKOs (b). The reduction in outgrowth was maintained on a laminin substrate in Gpsm2 cKOs (c). Data from three to six independent experiments are presented as whisker box plots (min/max) (n=number of growth cones). ***P<0.001 with unpaired Student's t-test. (d) Images from three time points (0, 15 and 30 min) of a time-lapse movie from 2 DIV control and Gpsm2 cKO hippocampal neurons plated on N-cadherin-coated glass and showing the difference in distance covered (dotted lines and double-headed arrows). Scale bar=10 μm. (e) Quantification of the average number of pauses (±s.e.m.) during a 30 min period of growth cones from control (n=31 neurons) compared with Gpsm2 cKO (n=51 neurons), from four independent experiments. The Gpsm2 cKO growth cones pause more than controls. ***P<0.001, *P<0.05 with unpaired Student's t-test. (f) Cumulated distance covered by control and Gpsm2 cKO growth cones over a 30 min period. Some pauses are indicated with blue arrows.
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f7: Gpsm2 mutation leads to hypoplasia of the corpus callosum and affects growth cone outgrowth.(a) Hematoxylin staining of coronal sections from P6 Gpsm2Emx1 control (upper panel) and cKO brains (lower panel) at a caudal levels. Note the abscence of corpus callossum (CC) in the cKO mouse (inset). Scale bar, 1 mm, n=4 independent experiments. (b,c) In cultured hippocampal neurons at DIV2, N-cadherin-dependent outgrowth was reduced in Gpsm2 cKOs, but not in Gnai3 cKOs (b). The reduction in outgrowth was maintained on a laminin substrate in Gpsm2 cKOs (c). Data from three to six independent experiments are presented as whisker box plots (min/max) (n=number of growth cones). ***P<0.001 with unpaired Student's t-test. (d) Images from three time points (0, 15 and 30 min) of a time-lapse movie from 2 DIV control and Gpsm2 cKO hippocampal neurons plated on N-cadherin-coated glass and showing the difference in distance covered (dotted lines and double-headed arrows). Scale bar=10 μm. (e) Quantification of the average number of pauses (±s.e.m.) during a 30 min period of growth cones from control (n=31 neurons) compared with Gpsm2 cKO (n=51 neurons), from four independent experiments. The Gpsm2 cKO growth cones pause more than controls. ***P<0.001, *P<0.05 with unpaired Student's t-test. (f) Cumulated distance covered by control and Gpsm2 cKO growth cones over a 30 min period. Some pauses are indicated with blue arrows.
Mentions: In addition to early-onset sensorineural deafness, patients with CMCS also display specific brain malformations on magnetic resonance images; hypoplasia of the CC being a hallmark of this pathology1. To evaluate the impact of Gpsm2 on CC development, we generated Emx1-Cre*Gpsm2 cKOs (Gpsm2Emx1), deleting Gpsm2 in the early and dorsal telencephalon. Analysis of brains from these cKOs confirmed the existence of a caudal CC agenesis (Fig. 7a), as reported in CMCS patients. CC defects can result from a disruption in neuronal progenitors or defects in axonal elongation and guidance, which are dependent upon the microtubule and actin cytoskeleton34. Neurite elongation is due to the motility of the growth cone driven in part by protrusive forces generated by actin polymerization at the leading edge3435 and the existence of a retrograde flow of actin resulting from a balance of filament polymerization and depolymerization, among other factors3637. We therefore hypothesized that actin dynamics could be impaired in mutant Gpsm2 neurons and affect outgrowth. Measures of growth cone locomotion on N-cadherin-coated substrates showed that outgrowth of Gpsm2 cKOs neurons was reduced by 37% compared with controls (control 0.97±0.04 μm min−1 versus cKO 0.61±0.02 μm min−1), whereas the speed of growth cones from Gnai3 cKOs was indistinguishable from controls (control 0.92±0.02 μm min−1; cKO 0.89±0.02 μm min−1) (Fig. 7b). This Gpsm2 cKO phenotype was also observed on a laminin-coated substrate, suggesting that the outgrowth reduction was not specific to an N-cadherin substrate (Fig. 7c). The number of pauses the growth cone made during the 30 min time-lapses on N-cadherin substrate was increased in Gpsm2 cKO neurons (Fig. 7d–f, Supplementary Movie 1).

View Article: PubMed Central - PubMed

ABSTRACT

Mutations in GPSM2 cause Chudley-McCullough syndrome (CMCS), an autosomal recessive neurological disorder characterized by early-onset sensorineural deafness and brain anomalies. Here, we show that mutation of the mouse orthologue of GPSM2 affects actin-rich stereocilia elongation in auditory and vestibular hair cells, causing deafness and balance defects. The G-protein subunit G&alpha;i3, a well-documented partner of Gpsm2, participates in the elongation process, and its absence also causes hearing deficits. We show that Gpsm2 defines an &sim;200&thinsp;nm nanodomain at the tips of stereocilia and this localization requires the presence of G&alpha;i3, myosin 15 and whirlin. Using single-molecule tracking, we report that loss of Gpsm2 leads to decreased outgrowth and a disruption of actin dynamics in neuronal growth cones. Our results elucidate the aetiology of CMCS and highlight a new molecular role for Gpsm2/G&alpha;i3 in the regulation of actin dynamics in epithelial and neuronal tissues.

No MeSH data available.


Related in: MedlinePlus