John J. Kelly

Mechanisms linking connexin mutations to human diseases

Ubiquitously expressed connexins are tetra-spanning transmembrane proteins that form intercellular gap junction channels or cell surface hemichannels. Connexins share similar topology but no sequence homology with mammalian pannexins and CALHM1 (calcium homeostasis modulator 1), which are also large-pore transmembrane channels. Of these three channel types, clinical evidence and gene sequence analysis to date have revealed that inherited human diseases are only associated with mutations in the connexin gene family. Connexin-linked diseases often present at birth or early in life and range from mild developmental abnormalities to severe organ failure such as hearing loss. Inherited connexin gene mutations can manifest as a disease by causing anomalies or defects in connexin oligomerization, folding, ability to pass quality control mechanisms or unexpected gain- or loss-of-function. This review provides examples of the way that various connexin gene mutations can cause disease via a wide range of molecular mechanisms. We also reflect on exciting strategies being explored in the connexin field and beyond with a view of translating their findings into potential connexin-disease therapeutics.

Panx1 Regulates Cellular Properties of Keratinocytes and Dermal Fibroblasts in Skin Development and Wound Healing

Pannexin1 (Panx1), a channel-forming glycoprotein is expressed in neonatal but not in aged mouse skin. Histological staining of Panx1 knockout (KO) mouse skin revealed a reduction in epidermal and dermal thickness and an increase in hypodermal adipose tissue. Following dorsal skin punch biopsies, mutant mice exhibited a significant delay in wound healing. Scratch wound and proliferation assays revealed that cultured keratinocytes from KO mice were more migratory, whereas dermal fibroblasts were more proliferative compared with controls. In addition, collagen gels populated with fibroblasts from KO mice exhibited significantly reduced contraction, comparable to WT fibroblasts treated with the Panx1 blocker, probenecid. KO fibroblasts did not increase α-smooth muscle actin expression in response to TGF-β, as is the case for differentiating WT myofibroblasts during wound contraction. We conclude that Panx1 controls cellular properties of keratinocytes and dermal fibroblasts during early stages of skin development and modulates wound repair upon injury.

A Germline Variant in the PANX1 Gene Has Reduced Channel Function and Is Associated with Multisystem Dysfunction

Pannexin1 (PANX1) is probably best understood as an ATP release channel involved in paracrine signaling. Given its ubiquitous expression, PANX1 pathogenic variants would be expected to lead to disorders involving multiple organ systems. Using whole exome sequencing, we discovered the first patient with a homozygous PANX1 variant (c.650G→A) resulting in an arginine to histidine substitution at position 217 (p.Arg217His). The 17-year-old female has intellectual disability, sensorineural hearing loss requiring bilateral cochlear implants, skeletal defects, including kyphoscoliosis, and primary ovarian failure. Her consanguineous parents are each heterozygous for this variant but are not affected by the multiorgan syndromes noted in the proband. Expression of the p.Arg217His mutant in HeLa, N2A, HEK293T, and Ad293 cells revealed normal PANX1 glycosylation and cell surface trafficking. Dye uptake, ATP release, and electrophysiological measurements revealed p.Arg217His to be a loss-of-function variant. Co-expression of the mutant with wild-type PANX1 suggested the mutant was not dominant-negative to PANX1 channel function. Collectively, we demonstrate a PANX1 missense change associated with human disease in the first report of a “PANX1-related disorder.”

Spinster Homolog 2 (Spns2) Deficiency Causes Early Onset Progressive Hearing Loss

Spinster homolog 2 (Spns2) acts as a Sphingosine-1-phosphate (S1P) transporter in zebrafish and mice, regulating heart development and lymphocyte trafficking respectively. S1P is a biologically active lysophospholipid with multiple roles in signalling. The mechanism of action of Spns2 is still elusive in mammals. Here, we report that Spns2-deficient mice rapidly lost auditory sensitivity and endocochlear potential (EP) from 2 to 3 weeks old. We found progressive degeneration of sensory hair cells in the organ of Corti, but the earliest defect was a decline in the EP, suggesting that dysfunction of the lateral wall was the primary lesion. In the lateral wall of adult mutants, we observed structural changes of marginal cell boundaries and of strial capillaries, and reduced expression of several key proteins involved in the generation of the EP (Kcnj10, Kcnq1, Gjb2 and Gjb6), but these changes were likely to be secondary. Permeability of the boundaries of the stria vascularis and of the strial capillaries appeared normal. We also found focal retinal degeneration and anomalies of retinal capillaries together with anterior eye defects in Spns2 mutant mice. Targeted inactivation of Spns2 in red blood cells, platelets, or lymphatic or vascular endothelial cells did not affect hearing, but targeted ablation of Spns2 in the cochlea using a Sox10-Cre allele produced a similar auditory phenotype to the original mutation, suggesting that local Spns2 expression is critical for hearing in mammals. These findings indicate that Spns2 is required for normal maintenance of the EP and hence for normal auditory function, and support a role for S1P signalling in hearing.

Mutations in Cx30 that are linked to skin disease and non-syndromic hearing loss exhibit several distinct cellular pathologies

ABSTRACT Connexin 30 (Cx30), a member of the large gap-junction protein family, plays a role in the homeostasis of the epidermis and inner ear through gap junctional intercellular communication (GJIC). Here, we investigate the underlying mechanisms of four autosomal dominant Cx30 gene mutations that are linked to hearing loss and/or various skin diseases. First, the T5M mutant linked to non-syndromic hearing loss formed functional gap junction channels and hemichannels, similar to wild-type Cx30. The loss-of-function V37E mutant associated with Clouston syndrome or keratitis-ichthyosis-deafness syndrome was retained in the endoplasmic reticulum and significantly induced apoptosis. The G59R mutant linked to the Vohwinkel and Bart-Pumphrey syndromes was retained primarily in the Golgi apparatus and exhibited loss of gap junction channel and hemichannel function but did not cause cell death. Lastly, the A88V mutant, which is linked to the development of Clouston syndrome, also significantly induced apoptosis but through an endoplasmic-reticulum-independent mechanism. Collectively, we discovered that four unique Cx30 mutants might cause disease through different mechanisms that also likely include their selective trans-dominant effects on coexpressed connexins, highlighting the overall complexity of connexin-linked diseases and the importance of GJIC in disease prevention.

Connexin30 mediated intercellular communication plays an essential role in epithelial repair in the cochlea.

Summary A role for connexin (Cx)30 in epithelial repair following injury was examined in the organ of Corti, the sensory epithelium of the cochlea. In this tissue, lesions caused by loss of the sensory hair cells are closed by the supporting cells that surround each one. Gap junctions in which Cx30 is the predominant connexin are large and numerous between supporting cells. In mice carrying a deletion in the gene (Gjb6) that encodes Cx30, the size and number of gap junction plaques, and the extent of dye transfer, between supporting cells was greatly reduced compared with normal animals. This corresponded with unique peculiarities of the lesion closure events during the progressive hair cell loss that occurs in these animals in comparison with other models of hair cell loss, whether acquired or as a result of a mutation. Only one, rather than all, of the supporting cells that contacted an individual dying hair closed the lesion, indicating disturbance of the co-ordination of cellular responses. The cell shape changes that the supporting cells normally undergo during repair of the organ of Corti did not occur. Also, there was disruption of the migratory activities that normally lead to the replacement of a columnar epithelium with a squamous-like one. These observations demonstrate a role for Cx30 and intercellular communication in regulating repair responses in an epithelial tissue.

The G60S Cx43 mutant enhances keratinocyte proliferation and differentiation

Transient knock‐down of the gap junction protein Cx43 by antisense and siRNA, or gap junction block with mimetic peptides, have been shown to enhance epidermal wound healing. However, patients with oculodentodigital dysplasia (ODDD) express mutant Cx43 that leads to a chronic reduction in gap junctional intercellular communication. To determine whether mutant Cx43 in keratinocytes would impact upon the wound healing process, we localized Cx43 in human and mouse skin tissue expressing mutant Cx43 and assessed the ability of primary keratinocytes derived from a mouse model of ODDD to proliferate, migrate and differentiate. In the epidermis from an ODDD patient and in the epidermis of mice expressing the G60S mutant or in keratinocytes obtained from mutant mice, Cx43 was frequently found within intracellular compartments and rarely localized to punctate sites of cell–cell apposition. Primary keratinocytes derived from G60S mutant mice proliferated faster but migrated similarly to keratinocytes derived from wild‐type control mice. Keratinocytes derived from mutant mice expressed abundant Cx43 and higher levels of involucrin and loricrin under low calcium conditions. However, after calcium‐induced differentiation, similar levels of Cx43, involucrin and loricrin were observed. Thus, we conclude that during wound healing, mutant Cx43 may enhance keratinocyte proliferation and promote early differentiation of keratinocytes.

Caprin-1 is a target of the deafness gene Pou4f3 and is recruited to stress granules in cochlear hair cells in response to ototoxic damage

The POU4 family of transcription factors are required for survival of specific cell types in different sensory systems. Pou4f3 is essential for the survival of auditory sensory hair cells and several mutations in human POU4F3 cause hearing loss. Thus, genes regulated by Pou4f3 are likely to be essential for hair cell survival. We performed a subtractive hybridisation screen in an inner-ear-derived cell line to find genes with differential expression in response to changes in Pou4f3 levels. The screen identified the stress-granule-associated protein Caprin-1 as being downregulated by Pou4f3. We demonstrated that this regulation occurs through the direct interaction of Pou4f3 with binding sites in the Caprin-1 5′ flanking sequence, and describe the expression pattern of Caprin-1 mRNA and protein in the cochlea. Moreover, we found Caprin-1-containing stress granules are induced in cochlear hair cells following aminoglycoside-induced damage. This is the first report of stress granule formation in mammalian hair cells and suggests that the formation of Caprin-1-containing stress granules is a key damage response to a clinically relevant ototoxic agent. Our results have implications for the understanding of aminoglycoside-induced hearing loss and provide further evidence that stress granule formation is a fundamental cellular stress response.

Manipulating Cx43 expression triggers gene reprogramming events in dermal fibroblasts from oculodentodigital dysplasia patients.

Oculodentodigital dysplasia (ODDD) is primarily an autosomal dominant disorder linked to over 70 GJA1 gene [connexin43 (Cx43)] mutations. For nearly a decade, our laboratory has been investigating the relationship between Cx43 and ODDD by expressing disease-linked mutants in reference cells, tissue-relevant cell lines, 3D organ cultures and by using genetically modified mouse models of human disease. Although salient features of Cx43 mutants have been revealed, these models do not necessarily reflect the complexity of the human context. To further overcome these limitations, we have acquired dermal fibroblasts from two ODDD-affected individuals harbouring D3N and V216L mutations in Cx43, along with familial controls. Using these ODDD patient dermal fibroblasts, which naturally produce less GJA1 gene product, along with RNAi and RNA activation (RNAa) approaches, we show that manipulating Cx43 expression triggers cellular gene reprogramming. Quantitative RT-PCR, Western blot and immunofluorescent analysis of ODDD patient fibroblasts show unusually high levels of extracellular matrix (ECM)-interacting proteins, including integrin α5β1, matrix metalloproteinases as well as secreted ECM proteins collagen-I and laminin. Cx43 knockdown in familial control cells produces similar effects on ECM expression, whereas Cx43 transcriptional up-regulation using RNAa decreases production of collagen-I. Interestingly, the enhanced levels of ECM-associated proteins in ODDD V216L fibroblasts is not only a consequence of increased ECM gene expression, but also due to an apparent deficit in collagen-I secretion which may further contribute to impaired collagen gel contraction in ODDD fibroblasts. These findings further illuminate the altered function of Cx43xa0in ODDD-affected individuals and highlight the impact of manipulating Cx43 expression in human cells.

Differential Effects of Pannexins on Noise-Induced Hearing Loss

Hearing loss, including noise-induced hearing loss, is highly prevalent and severely hinders an individuals quality of life, yet many of the mechanisms that cause hearing loss are unknown. The pannexin (Panx) channel proteins, Panx1 and Panx3, are regionally expressed in many cell types along the auditory pathway, and mice lacking Panx1 in specific cells of the inner ear exhibit hearing loss, suggesting a vital role for Panxs in hearing. We proposed that Panx1 and/or Panx3 null mice would exhibit severe hearing loss and increased susceptibility to noise-induced hearing loss. Using the auditory brainstem response, we surprisingly found that Panx1-/- and Panx3-/- mice did not harbor hearing or cochlear nerve deficits. Furthermore, while Panx1-/- mice displayed no protection against loud noise-induced hearing loss, Panx3-/- mice exhibited enhanced 16- and 24-kHz hearing recovery 7 days after a loud noise exposure (NE; 12u2005kHz tone, 115u2005dB sound pressure level, 1u2005h). Interestingly, Cx26, Cx30, Cx43, and Panx2 were up-regulated in Panx3-/- mice compared with wild-type and/or Panx1-/- mice, and assessment of the auditory tract revealed morphological changes in the middle ear bones of Panx3-/- mice. It is unclear if these changes alone are sufficient to provide protection against loud noise-induced hearing loss. Contrary to what we expected, these data suggest that Panx1 and Panx3 are not essential for baseline hearing in mice tested, but the therapeutic targeting of Panx3 may prove protective against mid-high-frequency hearing loss caused by loud NE.