The connexin30 A88V mutant reduces cochlear gap junction expression and confers long-term protection against hearing loss

Kelly, John J.; Abitbol, Julia M.; Hulme, Stephanie; Press, Eric R.; Laird, Dale W.; Allman, Brian L.

doi:10.1242/jcs.224097

Cited by 12 publications

(21 citation statements)

References 51 publications

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“…A similar increase in cell death has been observed for the A88V mutation in Cx30 [28]. Cx30 A88V also has reduced mRNA expression [28]. Whether this mutation triggers alternative splicing of the Cx30 mRNA in a manner analogous to what we report has not been investigated.…”

Section: Resultssupporting

confidence: 70%

Cx26 keratitis ichthyosis deafness syndrome mutations trigger alternative splicing of Cx26 to prevent expression and cause toxicityin vitro

2019

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The Cx26 mRNA has not been reported to undergo alternative splicing. In expressing a series of human keratitis ichthyosis deafness (KID) syndrome mutations of Cx26 (A88V, N14K and A40V), we found the production of a truncated mRNA product. These mutations, although not creating a cryptic splice site, appeared to activate a pre-existing cryptic splice site. The alternative splicing of the mutant Cx26 mRNA could be prevented by mutating the predicted 3′, 5′ splice sites and the branch point. The presence of a C-terminal fluorescent protein tag (mCherry or Clover) was necessary for this alternative splicing to occur. Strangely, Cx26 A88V could cause the alternative splicing of co-expressed WT Cx26—suggesting a trans effect. The alternative splicing of Cx26 A88V caused cell death, and this could be prevented by the 3′, 5′ and branch point mutations. Expression of the KID syndrome mutants could be rescued by combining them with removal of the 5′ splice site. We used this strategy to enable expression of Cx26 A40V-5′ and demonstrate that this KID syndrome mutation removed CO 2 sensitivity from the Cx26 hemichannel. This is the fourth KID syndrome mutation found to abolish the CO 2 -sensitivity of the Cx26 hemichannel, and suggests that the altered CO 2 -sensitivity could contribute to the pathology of this mutation. Future research on KID syndrome mutations should take care to avoid using a C-terminal tag to track cellular localization and expression or if this is unavoidable, combine this mutation with removal of the 5′ splice site.

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Section: Resultssupporting

confidence: 70%

Cx26 keratitis ichthyosis deafness syndrome mutations trigger alternative splicing of Cx26 to prevent expression and cause toxicityin vitro

2019

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“…HEI-OC1 cells that resemble a common progenitor to supporting cells and hair cells were derived from the epithelial region of the organ of Corti in mouse cochlear explants (Kalinec et al, 2003;Kalinec G. M. et al, 2016;Kelly et al, 2019). In keeping with most cell lines grown in culture, we observed that Cx43 was abundantly expressed in HEI-OC1 cells.…”

Section: Discussionsupporting

confidence: 73%

“…House Ear Institute-Organ of Corti 1 (HEI-OC1) cells were generously provided by Dr. Kalinec (House Ear Institute, Los Angeles, CA) (Kalinec et al, 2003;Kalinec G. M. et al, 2016;Kelly et al, 2019). HEI-OC1 cells were grown as we recently described (Abitbol et al, 2020).…”

Section: Cell Culture and Reagentsmentioning

confidence: 99%

“…Cells were stained with Hoechst (1:1000 diluted in distilled H 2 O, Cat# H3570, Molecular Probes) for 10 minutes to visualize the nuclei and coverslips were mounted using Airvol. C57BL/6 mouse cochleae were dissected and used in a cell lysate or cryosectioned, and immunolabeled as previously described (Kelly et al, 2019). Mouse usage for this purpose was approved by the Animal Care Committee at the University of Western Ontario.…”

Section: Western Blotting and Immunofluorescencementioning

confidence: 99%

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GJB2 Mutations Linked to Hearing Loss Exhibit Differential Trafficking and Functional Defects as Revealed in Cochlear-Relevant Cells

Beach

Abitbol

Allman

et al. 2020

Front. Cell Dev. Biol.

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GJB2 gene (that encodes Cx26) mutations are causal of hearing loss highlighting the importance of Cx26-based channel signaling amongst the supporting cells in the organ of Corti. While the majority of these GJB2 mutations are inherited in an autosomal recessive manner, others are inherited in an autosomal dominant manner and lead to syndromic hearing loss as well as skin diseases. To assess if common or divergent mechanisms are at the root of GJB2-linked hearing loss, we expressed several mutants in cochlear-relevant HEI-OC1 cells derived from the developing organ of Corti. Since supporting cells of the mature mammalian organ of Corti have negligible Cx43, but HEI-OC1 cells are rich in Cx43, we first used CRISPR-Cas9 to ablate endogenous Cx43, thus establishing a connexin-deficient platform for controlled reintroduction of hearing-relevant connexins and Cx26 mutants. We found three distinct outcomes and cellular phenotypes when hearing loss-linked Cx26 mutants were expressed in cochlear-relevant cells. The dominant syndromic Cx26 mutant N54K had trafficking defects and did not fully prevent wild-type Cx26 gap junction plaque formation but surprisingly formed gap junctions when co-expressed with Cx30. In contrast, the dominant syndromic S183F mutant formed gap junctions incapable of transferring dye and, as expected, co-localized in the same gap junctions as wild-type Cx26 and Cx30, but also gained the capacity to intermix with Cx43 within gap junctions. Both recessive non-syndromic Cx26 mutants (R32H and R184P) were retained in intracellular vesicles including early endosomes and did not co-localize with Cx30. As might be predicted, none of the Cx26 mutants prevented Cx43 gap junction plaque formation in Cx43-rich HEI-OC1 cells while Cx43-ablation had little effect on the expression of reference genes linked to auditory cell differentiation. We conclude from our studies in cochlear-relevant cells that the selected Cx26 mutants likely evoke hearing loss via three unique connexin defects that are independent of Cx43 status.

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“…[ 4 , 5 , 24 ] In line with this hypothesis, Cx30 A88V/A88V knock in mice (expressing Cx30 p.A88V in the homozygous state) exhibited enlarged SGs due to an increased number of sebocytes per SG, together with hyperproliferation of epidermal keratinocytes, as well as of cells forming the outermost layer of SGs [31] , which are mitotically active [32] . Recent work has highlighted also an atypical auditory phenotype and altered expression of cochlear gap junction channels in Cx30 A88V/A88V mice [ 33 , 34 ].…”

Section: Introductionmentioning

confidence: 99%

A potent antagonist antibody targeting connexin hemichannels alleviates Clouston syndrome symptoms in mutant mice

et al. 2020

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Background Numerous currently incurable human diseases have been causally linked to mutations in connexin (Cx) genes. In several instances, pathological mutations generate abnormally active Cx hemichannels, referred to also as “leaky” hemichannels. The goal of this study was to assay the in vivo efficacy of a potent antagonist antibody targeting Cx hemichannels. Methods We employed the antibody to treat Cx30 A88V/A88V adult mutant mice, the only available animal model of Clouston syndrome, a rare orphan disease caused by Cx30 p.A88V leaky hemichannels. To gain mechanistic insight into antibody action, we also performed patch clamp recordings, Ca 2+ imaging and ATP release assay in vitro . Findings Two weeks of antibody treatment sufficed to repress cell hyperproliferation in skin and reduce hypertrophic sebaceous glands (SGs) to wild type (wt) levels. These effects were obtained whether mutant mice were treated topically, by application of an antibody cream formulation, or systemically, by intraperitoneal antibody injection. Experiments with mouse primary keratinocytes and HaCaT cells revealed the antibody blocked Ca 2+ influx and diminished ATP release through leaky Cx30 p.A88V hemichannels. Interpretation Our results show anti-Cx antibody treatment was effective in vivo and sufficient to counteract the effects of pathological connexin expression in Cx30 A88V/A88V mice. In vitro experiments suggest antibodies gained control over leaky hemichannels and contributed to restoring epidermal homeostasis. Therefore, regulating cell physiology by antibodies targeting the extracellular domain of Cxs may enforce an entirely new therapeutic strategy. These findings support the further development of antibodies as drugs to address unmet medical needs for Cx-related diseases. Fund Fondazione Telethon, GGP19148; University of Padova, SID/BIRD187130; Consiglio Nazionale delle Ricerche, DSB.AD008.370.003\TERABIO-IBCN; National Science Foundation of China, 31770776; Science and Technology Commission of Shanghai Municipality, 16DZ1910200.

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The connexin30 A88V mutant reduces cochlear gap junction expression and confers long-term protection against hearing loss

Cited by 12 publications

References 51 publications

Cx26 keratitis ichthyosis deafness syndrome mutations trigger alternative splicing of Cx26 to prevent expression and cause toxicityin vitro

Cx26 keratitis ichthyosis deafness syndrome mutations trigger alternative splicing of Cx26 to prevent expression and cause toxicityin vitro

GJB2 Mutations Linked to Hearing Loss Exhibit Differential Trafficking and Functional Defects as Revealed in Cochlear-Relevant Cells

A potent antagonist antibody targeting connexin hemichannels alleviates Clouston syndrome symptoms in mutant mice

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