Connexin 50 Influences the Physiological Optics of the In Vivo Mouse Lens.

Purpose: The purpose of this study was to utilize multi-parametric magnetic resonance imaging (MRI) to investigate in vivo age-related changes in the physiology and optics of mouse lenses where Connexin 50 has been deleted (Cx50KO) or replaced by Connexin 46 (Cx50KI46).

Methods: The lenses of transgenic Cx50KO and Cx50KI46 mice were imaged between 3 weeks and 6 months of age using a 7T MRI. Measurements of lens geometry, the T2 (water-bound protein ratios), the refractive index (n), and T1 (free water content) values were calculated by processing the acquired images.

Single-Cell RNA Sequencing Analysis of the Early Postnatal Mouse Lens Epithelium.

Purpose: The lens epithelium maintains the overall health of the organ. We used single-cell RNA sequencing (scRNA-seq) technology to assess transcriptional heterogeneity between cells in the postnatal day 2 (P2) epithelium and identify distinct epithelial cell subtypes. Analysis of these data was used to better understand lens growth, differentiation, and homeostasis on P2.

Age-Dependent Changes in the Water Content and Optical Power of the In Vivo Mouse Lens Revealed by Multi-Parametric MRI and Optical Modeling.

Purpose: The purpose of this study was to utilize in vivo magnetic resonance imaging (MRI) and optical modeling to investigate how changes in water transport, lens curvature, and gradient refractive index (GRIN) alter the power of the mouse lens as a function of age.

Methods: Lenses of male C57BL/6 wild-type mice aged between 3 weeks and 12 months (N = 4 mice per age group) were imaged using a 7T MRI scanner. Measurements of lens shape and the distribution of T2 (water-bound protein ratios) and T1 (free water content) values were extracted from MRI images.

Antibody gene transfer treatment drastically improves epidermal pathology in a keratitis ichthyosis deafness syndrome model using male mice.

Background: Keratitis ichthyosis deafness (KID) syndrome is a rare disorder caused by hemichannel (HC) activating gain-of-function mutations in the GJB2 gene encoding connexin (Cx) 26, for which there is no cure, or current treatments based upon the mechanism of disease causation.

Methods: We applied Adeno Associated Virus (AAV) mediated mAb gene transfer (AAVmAb) to treat the epidermal features of KID syndrome with a well-characterized HC blocking antibody using male mice of a murine model that replicates the skin pathology of the human disease.

Findings: We demonstrate that in vivo AAVmAb treatment significantly reduced the size and thickness of KID lesions, in addition to blocking activity of mutant HCs in the epidermis in vivo.

Double Deletion of PI3K and PTEN Modifies Lens Postnatal Growth and Homeostasis.

We have previously shown that the conditional deletion of either the p110α catalytic subunit of phosphatidylinositol 3-kinase (PI3K), or its opposing phosphatase, phosphatase and tensin homolog (PTEN), had distinct effects on lens growth and homeostasis. The deletion of p110α reduced the levels of phosphorylated Akt and equatorial epithelial cell proliferation, and resulted in smaller transparent lenses in adult mice. The deletion of PTEN increased levels of phosphorylated Akt, altered lens sodium transport, and caused lens rupture and cataract.

Physiological Mechanisms Regulating Lens Transport.

The transparency and refractive properties of the lens are maintained by the cellular physiology provided by an internal microcirculation system that utilizes spatial differences in ion channels, transporters and gap junctions to establish standing electrochemical and hydrostatic pressure gradients that drive the transport of ions, water and nutrients through this avascular tissue. Aging has negative effects on lens transport, degrading ion and water homeostasis, and producing changes in lens water content. This alters the properties of the lens, causing changes in optical quality and accommodative amplitude that initially result in presbyopia in middle age and ultimately manifest as cataract in the elderly.

Connexin hemichannel inhibition ameliorates epidermal pathology in a mouse model of keratitis ichthyosis deafness syndrome.

Mutations in five different genes encoding connexin channels cause eleven clinically defined human skin diseases. Keratitis ichthyosis deafness (KID) syndrome is caused by point mutations in the GJB2 gene encoding Connexin 26 (Cx26) which result in aberrant activation of connexin hemichannels. KID syndrome has no cure and is associated with bilateral hearing loss, blinding keratitis, palmoplantar keratoderma, ichthyosiform erythroderma and a high incidence of childhood mortality.

Signaling Between TRPV1/TRPV4 and Intracellular Hydrostatic Pressure in the Mouse Lens.

Purpose: The lens uses feedback to maintain zero pressure in its surface cells. Positive pressures are detected by transient receptor potential vanilloid (TRPV4), which initiates a cascade that reduces surface cell osmolarity. The first step is opening of gap junction hemichannels.

Multi-parametric MRI of the physiology and optics of the in-vivo mouse lens.

The optics of the ocular lens are determined by its geometry (shape and volume) and its inherent gradient of refractive index (water to protein ratio), which are in turn maintained by unique cellular physiology known as the lens internal microcirculation system. Previously, magnetic resonance imaging (MRI) has been used on ex vivo organ cultured bovine lenses to show that pharmacological perturbations to this microcirculation system disrupt ionic and fluid homeostasis and overall lens optics. In this study, we have optimised in vivo MRI protocols for use on wild-type and transgenic mouse models so that the effects of genetically perturbing the lens microcirculation system on lens properties can be studied.

The Ciliary Muscle and Zonules of Zinn Modulate Lens Intracellular Hydrostatic Pressure Through Transient Receptor Potential Vanilloid Channels.

Purpose: Lenses have an intracellular hydrostatic pressure gradient to drive fluid from central fiber cells to surface epithelial cells. Pressure is regulated by a feedback control system that relies on transient receptor potential vanilloid (TRPV)1 and TRPV4 channels. The ciliary muscle transmits tension to the lens through the zonules of Zinn.

Connexin43 mutations linked to skin disease have augmented hemichannel activity.

Mutations in the gene (GJA1) encoding connexin43 (Cx43) are responsible for several rare genetic disorders, including non-syndromic skin-limited diseases. Here we used two different functional expression systems to characterize three Cx43 mutations linked to palmoplantar keratoderma and congenital alopecia-1, erythrokeratodermia variabilis et progressiva, or inflammatory linear verrucous epidermal nevus. In HeLa cells and Xenopus oocytes, we show that Cx43-G8V, Cx43-A44V and Cx43-E227D all formed functional gap junction channels with the same efficiency as wild-type Cx43, with normal voltage gating and a unitary conductance of ~110 pS.

The Phosphoinosotide 3-Kinase Catalytic Subunit p110α is Required for Normal Lens Growth.

Purpose: Signal transduction pathways influence lens growth, but little is known about the role(s) of the class 1A phosphoinositide 3-kinases (PI3Ks). To further investigate how signaling regulates lens growth, we generated and characterized mice in which the p110α and p110β catalytic subunits of PI3K were conditionally deleted in the mouse lens.

Methods: Floxed alleles of the catalytic subunits of PI3K were conditionally deleted in the lens by using MLR10-cre transgenic mice.

Connexin channels in congenital skin disorders.

Gap junctions and hemichannels comprised of connexins influence epidermal proliferation and differentiation. Significant advances in our understanding of the functional role of connexins in the skin have been made by studying the diseases caused by connexin mutations. Eleven clinically defined cutaneous disorders with an overlapping spectrum of phenotypes are caused by mutations in five different connexin genes, highlighting that disease presentation must be deciphered with an understanding of how connexin functions are affected.

Connexin 46 (cx46) gap junctions provide a pathway for the delivery of glutathione to the lens nucleus.

Maintenance of adequate levels of glutathione (GSH) in the lens nucleus is critical for protection of lens proteins from the effects of oxidative stress and for lens transparency. How GSH is transported to the nucleus is unknown. We show that GSH diffuses to the nucleus from the outer cortex, where a high concentration of the anti-oxidant is established by synthesis or uptake, via the network of gap junctions.

AKT activation promotes PTEN hamartoma tumor syndrome-associated cataract development.

Mutations in the human phosphatase and tensin homolog (PTEN) gene cause PTEN hamartoma tumor syndrome (PHTS), which includes cataract development among its diverse clinical pathologies. Currently, it is not known whether cataract formation in PHTS patients is secondary to other systemic problems, or the result of the loss of a critical function of PTEN within the lens. We generated a mouse line with a lens-specific deletion of Pten (PTEN KO) and identified a regulatory function for PTEN in lens ion transport.

The Cx26-G45E mutation displays increased hemichannel activity in a mouse model of the lethal form of keratitis-ichthyosis-deafness syndrome.

Mutations in the GJB2 gene (Cx26) cause deafness in humans. Most are loss-of-function mutations and cause nonsyndromic deafness. Some mutations produce a gain of function and cause syndromic deafness associated with skin disorders, such as keratitis-ichthyosis-deafness syndrome (KIDS).

The cataract causing Cx50-S50P mutant inhibits Cx43 and intercellular communication in the lens epithelium.

Mutations in Connexin50 (Cx50) cause cataracts in both humans and mice. The mechanism(s) behind how mutated connexins lead to a variety of cataracts have yet to be fully elucidated. Here, we tested whether the cataract inducing Cx50-S50P mutant interacts with wild-type Connexin43 (Cx43) to form mixed channels with attenuated function.

Interaction between Connexin50 and mitogen-activated protein kinase signaling in lens homeostasis.

Both connexins and signal transduction pathways have been independently shown to play critical roles in lens homeostasis, but little is known about potential cooperation between these two intercellular communication systems. To investigate whether growth factor signaling and gap junctional communication interact during the development of lens homeostasis, we examined the effect of mitogen-activated protein kinase (MAPK) signaling on coupling mediated by specific lens connexins by using a combination of in vitro and in vivo assays. Activation of MAPK signaling pathways significantly increased coupling provided by Cx50, but not Cx46, in paired Xenopus laevis oocytes in vitro, as well as between freshly isolated lens cells in vivo.

Optimal lens epithelial cell proliferation is dependent on the connexin isoform providing gap junctional coupling.

Purpose: Gap junctions between epithelial cells are essential for normal lens growth. In mice, knockout of Cx50 or targeted replacement of Cx50 with Cx46 (knockin) caused smaller lenses because of decreased epithelial cell proliferation. However, it remains unclear whether Cx50 functionally contributes to lens epithelial coupling during maximal proliferation on postnatal day 2 (P2) and P3.

Mefloquine effects on the lens suggest cooperative gating of gap junction channels.

Mefloquine (MFQ) selectively blocks exogenously expressed gap junction channels composed of Cx50 but not Cx46. The purpose of the current study was to evaluate MFQ effects on wild-type (WT) mouse lenses that express both Cx50 and Cx46 in their outer shell of differentiating fibers (DFs). Lenses in which Cx46 was knocked into both Cx50 alleles (KI) were used as controls; MFQ had no effect on coupling in these lenses.

Lens gap junctional coupling is modulated by connexin identity and the locus of gene expression.

Purpose: To investigate the effects of reducing connexin (Cx) diversity in the lens when the amount of connexin protein is nearly constant.

Methods: Lenses in which the Cx50 coding region was replaced by that of Cx46 (knockin [KI]), were compared with wild type (WT) and Cx50-knockout (KO) lenses. Gap junctional conductance (G(j)), and membrane conductance were evaluated by using frequency domain impedance of intact lenses.

Connexin50 is essential for normal postnatal lens cell proliferation.

Purpose: Connexin50 (Cx50) is absolutely essential for normal postnatal lens growth. Deletion of Cx50 or replacement with Cx46 by knockin resulted in smaller lenses containing fewer cells. To determine why Cx50-deficient lenses fail to grow normally, cell proliferation was assayed during the period of growth failure.

Dominant cataracts result from incongruous mixing of wild-type lens connexins.

Gap junctions are composed of proteins called connexins (Cx) and facilitate both ionic and biochemical modes of intercellular communication. In the lens, Cx46 and Cx50 provide the gap junctional coupling needed for homeostasis and growth. In mice, deletion of Cx46 produced severe cataracts, whereas knockout of Cx50 resulted in significantly reduced lens growth and milder cataracts.

Genetic background influences cataractogenesis, but not lens growth deficiency, in Cx50-knockout mice.

Purpose: Deletion of connexin (Cx)50 produces microphthalmia with nuclear cataracts. To determine whether these two traits are influenced by genetic background and are dependent on each other, mice carrying the Cx50 deletion in two different strains were generated, and the growth defect and severity of cataracts were analyzed.

Methods: Cx50-knockout mice were generated in the 129S6 strain, and back-crossed into the C57BL/6J genetic background.