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Gipc3 mutations associated with audiogenic seizures and sensorineural hearing loss in mouse and human.

Charizopoulou N, Lelli A, Schraders M, Ray K, Hildebrand MS, Ramesh A, Srisailapathy CR, Oostrik J, Admiraal RJ, Neely HR, Latoche JR, Smith RJ, Northup JK, Kremer H, Holt JR, Noben-Trauth K - Nat Commun (2011)

Bottom Line: A missense mutation in the PDZ domain has an attenuating effect on mechanotransduction and the acquisition of mature inner hair cell potassium currents.The Gipc3(343A) allele disrupts the structure of the stereocilia bundle and affects long-term function of auditory hair cells and spiral ganglion neurons.Our study suggests a pivotal role of Gipc3 in acoustic signal acquisition and propagation in cochlear hair cells.

View Article: PubMed Central - PubMed

Affiliation: Section on Neurogenetics, Laboratory of Molecular Biology, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Rockville, Maryland 20850, USA.

ABSTRACT
Sensorineural hearing loss affects the quality of life and communication of millions of people, but the underlying molecular mechanisms remain elusive. Here, we identify mutations in Gipc3 underlying progressive sensorineural hearing loss (age-related hearing loss 5, ahl5) and audiogenic seizures (juvenile audiogenic monogenic seizure 1, jams1) in mice and autosomal recessive deafness DFNB15 and DFNB95 in humans. Gipc3 localizes to inner ear sensory hair cells and spiral ganglion. A missense mutation in the PDZ domain has an attenuating effect on mechanotransduction and the acquisition of mature inner hair cell potassium currents. Magnitude and temporal progression of wave I amplitude of afferent neurons correlate with susceptibility and resistance to audiogenic seizures. The Gipc3(343A) allele disrupts the structure of the stereocilia bundle and affects long-term function of auditory hair cells and spiral ganglion neurons. Our study suggests a pivotal role of Gipc3 in acoustic signal acquisition and propagation in cochlear hair cells.

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Protein blot analysis and immunolocalization of Gipc3.(a) Western blot analysis of Gipc3343G transfected (+) and untransfected (−) HEK293T cell lysates using Gipc3-specific polyclonal antiserum Pb867 detecting a single ∼40 kDa band (black arrow). (b) Immunoblot analysis of protein extracts from cochlea and testis from BLSW and C3HeB/FeJ (C3 H) mice with anti-Gipc3 antiserum Pb867. Lysates from HeLa cells transfected with the wild-type Gipc3343G construct were included as control. The ∼40 kDa band (black arrow) was detected in both tissues from C3HeB/FeJ wild-type mice. The similar ∼40 kDa band was detected with reduced intensity in mutant BLSW cochlea and testis. The ∼37 kDa band (arrow head) and ∼29 kDa band (dot, presumably nonspecific) are indicated. (c) Quantitative analysis of wild-type Gipc3(115Gly) and mutant Gipc3(115Arg) expression in lysates from transiently transfected HEK293T cells with varying amounts of protein loaded (10 and 30 μg) for wild-type Gipc3343G and mutant Gipc3343A. Protein blots were probed with the anti-Gipc3 antiserum Pb867and anti-α-actin antibody. (d) Densitometric analysis of Gipc3 expression from wild-type Gipc3(115Gly) and mutant Gipc3(115Arg)-transfected HEK293T cells. The data are represented as mean±s.e.m., **P<0.01 (n=6, t-test). Confocal images of cryosections of organ of Corti (e) and spiral ganglion (f) stained with Pb867 antibody (red) demonstrating Gipc3 localization in IHCs (arrow head) and OHCs (arrow) and spiral ganglion (arrow). Confocal images of organ of Corti of C3HeB/FeJ (g, h, j) and BLSW (i, k, l) demonstrating Gipc3 (g–i) and Vglut3 (k) and myosin VI (l) localization; (i–l) counterstained with phalloidin (green); arrows point to positive staining. (e–k) Counterstained with 4,6-diamidino-2-phenylindole (blue). (l) No Pb867-staining is seen in stereocilia of IHC (arrow head); scale bar, 5 μm (e–l).
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f6: Protein blot analysis and immunolocalization of Gipc3.(a) Western blot analysis of Gipc3343G transfected (+) and untransfected (−) HEK293T cell lysates using Gipc3-specific polyclonal antiserum Pb867 detecting a single ∼40 kDa band (black arrow). (b) Immunoblot analysis of protein extracts from cochlea and testis from BLSW and C3HeB/FeJ (C3 H) mice with anti-Gipc3 antiserum Pb867. Lysates from HeLa cells transfected with the wild-type Gipc3343G construct were included as control. The ∼40 kDa band (black arrow) was detected in both tissues from C3HeB/FeJ wild-type mice. The similar ∼40 kDa band was detected with reduced intensity in mutant BLSW cochlea and testis. The ∼37 kDa band (arrow head) and ∼29 kDa band (dot, presumably nonspecific) are indicated. (c) Quantitative analysis of wild-type Gipc3(115Gly) and mutant Gipc3(115Arg) expression in lysates from transiently transfected HEK293T cells with varying amounts of protein loaded (10 and 30 μg) for wild-type Gipc3343G and mutant Gipc3343A. Protein blots were probed with the anti-Gipc3 antiserum Pb867and anti-α-actin antibody. (d) Densitometric analysis of Gipc3 expression from wild-type Gipc3(115Gly) and mutant Gipc3(115Arg)-transfected HEK293T cells. The data are represented as mean±s.e.m., **P<0.01 (n=6, t-test). Confocal images of cryosections of organ of Corti (e) and spiral ganglion (f) stained with Pb867 antibody (red) demonstrating Gipc3 localization in IHCs (arrow head) and OHCs (arrow) and spiral ganglion (arrow). Confocal images of organ of Corti of C3HeB/FeJ (g, h, j) and BLSW (i, k, l) demonstrating Gipc3 (g–i) and Vglut3 (k) and myosin VI (l) localization; (i–l) counterstained with phalloidin (green); arrows point to positive staining. (e–k) Counterstained with 4,6-diamidino-2-phenylindole (blue). (l) No Pb867-staining is seen in stereocilia of IHC (arrow head); scale bar, 5 μm (e–l).

Mentions: We reasoned that the Gly115Arg mutation might alter the expression or stability of Gipc3. To test this hypothesis, we probed western blots with polyclonal antisera raised against Gipc3-specific peptides. In protein extracts derived from HEK293T cells transiently expressing wild-type Gipc3, the Pb867, Pb869 and Pb877 antisera detected a single ∼40 kDa immunoreactive band (Fig. 6a and Supplementary Fig. S2). When we probed western blots of cochlea and testis protein extracts from BLSW and C3HeB/FeJ we detected a reduction in intensity of the ∼40 kDa band in BLSW cochlea extracts by 82±7% (n=3) and in testis extracts by 42±9% (n=3) compared with wild-type C3HeB/FeJ extracts (Fig. 6b). As an additional test of the expression level differences, we generated a mutant Gipc3343A cDNA construct for expression in HEK293T cells. Quantification, after normalizing against actin expression, revealed a reduction in expression levels by 69% of the mutant Gipc3(115Arg) protein compared with that for wild-type Gipc3(115Gly) (Fig. 6c,d). TaqMan qPCR analyses revealed no significant differences in Gipc3 mRNA expression in HEK293T cells transfected with wild-type (Gipc3343G) or mutant (Gipc3343A) construct (data not shown). In addition to the ∼40 kDa band, Pb867 recognizes a ∼37 kDa band in both cochlea and testis extracts. Both bands can be blocked by the Gipc3-specific peptide used for generating the Pb867 antibody and are also detected by the Pb869/Pb877 antiserum (Supplementary Fig. S2). In addition, Pb867 is specific for Gipc3, showing strong reactivity with human GIPC3 and only faint cross reactivity with mouse Gipc1 and Gipc2 (Supplementary Fig. S2). Thus, the ∼37 kDa band may represent either an alternate isoform or partially cleaved product of the ∼40 kDa protein. Together, these data indicate that the Gly115Arg substitution causes a reduction of the 40 kDa Gipc3 protein in BLSW cochleae.


Gipc3 mutations associated with audiogenic seizures and sensorineural hearing loss in mouse and human.

Charizopoulou N, Lelli A, Schraders M, Ray K, Hildebrand MS, Ramesh A, Srisailapathy CR, Oostrik J, Admiraal RJ, Neely HR, Latoche JR, Smith RJ, Northup JK, Kremer H, Holt JR, Noben-Trauth K - Nat Commun (2011)

Protein blot analysis and immunolocalization of Gipc3.(a) Western blot analysis of Gipc3343G transfected (+) and untransfected (−) HEK293T cell lysates using Gipc3-specific polyclonal antiserum Pb867 detecting a single ∼40 kDa band (black arrow). (b) Immunoblot analysis of protein extracts from cochlea and testis from BLSW and C3HeB/FeJ (C3 H) mice with anti-Gipc3 antiserum Pb867. Lysates from HeLa cells transfected with the wild-type Gipc3343G construct were included as control. The ∼40 kDa band (black arrow) was detected in both tissues from C3HeB/FeJ wild-type mice. The similar ∼40 kDa band was detected with reduced intensity in mutant BLSW cochlea and testis. The ∼37 kDa band (arrow head) and ∼29 kDa band (dot, presumably nonspecific) are indicated. (c) Quantitative analysis of wild-type Gipc3(115Gly) and mutant Gipc3(115Arg) expression in lysates from transiently transfected HEK293T cells with varying amounts of protein loaded (10 and 30 μg) for wild-type Gipc3343G and mutant Gipc3343A. Protein blots were probed with the anti-Gipc3 antiserum Pb867and anti-α-actin antibody. (d) Densitometric analysis of Gipc3 expression from wild-type Gipc3(115Gly) and mutant Gipc3(115Arg)-transfected HEK293T cells. The data are represented as mean±s.e.m., **P<0.01 (n=6, t-test). Confocal images of cryosections of organ of Corti (e) and spiral ganglion (f) stained with Pb867 antibody (red) demonstrating Gipc3 localization in IHCs (arrow head) and OHCs (arrow) and spiral ganglion (arrow). Confocal images of organ of Corti of C3HeB/FeJ (g, h, j) and BLSW (i, k, l) demonstrating Gipc3 (g–i) and Vglut3 (k) and myosin VI (l) localization; (i–l) counterstained with phalloidin (green); arrows point to positive staining. (e–k) Counterstained with 4,6-diamidino-2-phenylindole (blue). (l) No Pb867-staining is seen in stereocilia of IHC (arrow head); scale bar, 5 μm (e–l).
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f6: Protein blot analysis and immunolocalization of Gipc3.(a) Western blot analysis of Gipc3343G transfected (+) and untransfected (−) HEK293T cell lysates using Gipc3-specific polyclonal antiserum Pb867 detecting a single ∼40 kDa band (black arrow). (b) Immunoblot analysis of protein extracts from cochlea and testis from BLSW and C3HeB/FeJ (C3 H) mice with anti-Gipc3 antiserum Pb867. Lysates from HeLa cells transfected with the wild-type Gipc3343G construct were included as control. The ∼40 kDa band (black arrow) was detected in both tissues from C3HeB/FeJ wild-type mice. The similar ∼40 kDa band was detected with reduced intensity in mutant BLSW cochlea and testis. The ∼37 kDa band (arrow head) and ∼29 kDa band (dot, presumably nonspecific) are indicated. (c) Quantitative analysis of wild-type Gipc3(115Gly) and mutant Gipc3(115Arg) expression in lysates from transiently transfected HEK293T cells with varying amounts of protein loaded (10 and 30 μg) for wild-type Gipc3343G and mutant Gipc3343A. Protein blots were probed with the anti-Gipc3 antiserum Pb867and anti-α-actin antibody. (d) Densitometric analysis of Gipc3 expression from wild-type Gipc3(115Gly) and mutant Gipc3(115Arg)-transfected HEK293T cells. The data are represented as mean±s.e.m., **P<0.01 (n=6, t-test). Confocal images of cryosections of organ of Corti (e) and spiral ganglion (f) stained with Pb867 antibody (red) demonstrating Gipc3 localization in IHCs (arrow head) and OHCs (arrow) and spiral ganglion (arrow). Confocal images of organ of Corti of C3HeB/FeJ (g, h, j) and BLSW (i, k, l) demonstrating Gipc3 (g–i) and Vglut3 (k) and myosin VI (l) localization; (i–l) counterstained with phalloidin (green); arrows point to positive staining. (e–k) Counterstained with 4,6-diamidino-2-phenylindole (blue). (l) No Pb867-staining is seen in stereocilia of IHC (arrow head); scale bar, 5 μm (e–l).
Mentions: We reasoned that the Gly115Arg mutation might alter the expression or stability of Gipc3. To test this hypothesis, we probed western blots with polyclonal antisera raised against Gipc3-specific peptides. In protein extracts derived from HEK293T cells transiently expressing wild-type Gipc3, the Pb867, Pb869 and Pb877 antisera detected a single ∼40 kDa immunoreactive band (Fig. 6a and Supplementary Fig. S2). When we probed western blots of cochlea and testis protein extracts from BLSW and C3HeB/FeJ we detected a reduction in intensity of the ∼40 kDa band in BLSW cochlea extracts by 82±7% (n=3) and in testis extracts by 42±9% (n=3) compared with wild-type C3HeB/FeJ extracts (Fig. 6b). As an additional test of the expression level differences, we generated a mutant Gipc3343A cDNA construct for expression in HEK293T cells. Quantification, after normalizing against actin expression, revealed a reduction in expression levels by 69% of the mutant Gipc3(115Arg) protein compared with that for wild-type Gipc3(115Gly) (Fig. 6c,d). TaqMan qPCR analyses revealed no significant differences in Gipc3 mRNA expression in HEK293T cells transfected with wild-type (Gipc3343G) or mutant (Gipc3343A) construct (data not shown). In addition to the ∼40 kDa band, Pb867 recognizes a ∼37 kDa band in both cochlea and testis extracts. Both bands can be blocked by the Gipc3-specific peptide used for generating the Pb867 antibody and are also detected by the Pb869/Pb877 antiserum (Supplementary Fig. S2). In addition, Pb867 is specific for Gipc3, showing strong reactivity with human GIPC3 and only faint cross reactivity with mouse Gipc1 and Gipc2 (Supplementary Fig. S2). Thus, the ∼37 kDa band may represent either an alternate isoform or partially cleaved product of the ∼40 kDa protein. Together, these data indicate that the Gly115Arg substitution causes a reduction of the 40 kDa Gipc3 protein in BLSW cochleae.

Bottom Line: A missense mutation in the PDZ domain has an attenuating effect on mechanotransduction and the acquisition of mature inner hair cell potassium currents.The Gipc3(343A) allele disrupts the structure of the stereocilia bundle and affects long-term function of auditory hair cells and spiral ganglion neurons.Our study suggests a pivotal role of Gipc3 in acoustic signal acquisition and propagation in cochlear hair cells.

View Article: PubMed Central - PubMed

Affiliation: Section on Neurogenetics, Laboratory of Molecular Biology, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Rockville, Maryland 20850, USA.

ABSTRACT
Sensorineural hearing loss affects the quality of life and communication of millions of people, but the underlying molecular mechanisms remain elusive. Here, we identify mutations in Gipc3 underlying progressive sensorineural hearing loss (age-related hearing loss 5, ahl5) and audiogenic seizures (juvenile audiogenic monogenic seizure 1, jams1) in mice and autosomal recessive deafness DFNB15 and DFNB95 in humans. Gipc3 localizes to inner ear sensory hair cells and spiral ganglion. A missense mutation in the PDZ domain has an attenuating effect on mechanotransduction and the acquisition of mature inner hair cell potassium currents. Magnitude and temporal progression of wave I amplitude of afferent neurons correlate with susceptibility and resistance to audiogenic seizures. The Gipc3(343A) allele disrupts the structure of the stereocilia bundle and affects long-term function of auditory hair cells and spiral ganglion neurons. Our study suggests a pivotal role of Gipc3 in acoustic signal acquisition and propagation in cochlear hair cells.

Show MeSH
Related in: MedlinePlus