Myopia Alters the Structural Organization of the Retinal Vasculature, GFAP-Positive Glia, and Ganglion Cell Layer Thickness.

To describe the effect of myopic eye growth on the structure and distribution of astrocytes, vasculature, and retinal nerve fiber layer thickness, which are critical for inner retinal tissue homeostasis and survival. Astrocyte and capillary distribution, retinal nerve fiber (RNFL), and ganglion cell layer (GCL) thicknesses were assessed using immunochemistry and spectral domain optical coherence tomography on eleven retinas of juvenile common marmosets (Callithrix Jacchus), six of which were induced with lens-induced myopia (refraction, Rx: -7.01 ± 1.8D). Five untreated age-matched juvenile marmoset retinas were used as controls (Rx: -0.74 ± 0.4D). Untreated marmoset eyes grew normally, their RNFL thickened and their astrocyte numbers were associated with RNFL thickness. Marmosets with induced myopia did not show this trend and, on the contrary, had reduced astrocyte numbers, increased GFAP-immunopositive staining, thinner RNFL, lower peripheral capillary branching, and increased numbers of string vessels. The myopic changes in retinal astrocytes, vasculature, and retinal nerve fiber layer thickness suggest a reorganization of the astrocyte and vascular templates during myopia development and progression. Whether these adaptations are beneficial or harmful to the retina remains to be investigated.

Suppression of connexin 43 phosphorylation promotes astrocyte survival and vascular regeneration in proliferative retinopathy.

Degeneration of retinal astrocytes precedes hypoxia-driven pathologic neovascularization and vascular leakage in ischemic retinopathies. However, the molecular events that underlie astrocyte loss remain unclear. Astrocytes abundantly express connexin 43 (Cx43), a transmembrane protein that forms gap junction (GJ) channels and hemichannels. Cx channels can transfer toxic signals from dying cells to healthy neighbors under pathologic conditions. Here we show that Cx43 plays a critical role in astrocyte apoptosis and the resulting preretinal neovascularization in a mouse model of oxygen-induced retinopathy. Opening of Cx43 hemichannels was not observed following hypoxia. In contrast, GJ coupling between astrocytes increased, which could lead to amplification of injury. Accordingly, conditional deletion of Cx43 maintained a higher density of astrocytes in the hypoxic retina. We also identify a role for Cx43 phosphorylation in mediating these processes. Increased coupling in response to hypoxia is due to phosphorylation of Cx43 by casein kinase 1δ (CK1δ). Suppression of this phosphorylation using an inhibitor of CK1δ or in site-specific phosphorylation-deficient mice similarly protected astrocytes from hypoxic damage. Rescue of astrocytes led to restoration of a functional retinal vasculature and lowered the hypoxic burden, thereby curtailing neovascularization and neuroretinal dysfunction. We also find that absence of astrocytic Cx43 does not affect developmental angiogenesis or neuronal function in normoxic retinas. Our in vivo work directly links phosphorylation of Cx43 to astrocytic coupling and apoptosis and ultimately to vascular regeneration in retinal ischemia. This study reveals that targeting Cx43 phosphorylation in astrocytes is a potential direction for the treatment of proliferative retinopathies.

Altered inhibition of Cx26 hemichannels by pH and Zn2+ in the A40V mutation associated with keratitis-ichthyosis-deafness syndrome.

Excessive opening of undocked Cx26 hemichannels in the plasma membrane is associated with disease pathogenesis in keratitis-ichthyosis-deafness (KID) syndrome. Thus far, excessive opening of KID mutant hemichannels has been attributed, almost solely, to aberrant inhibition by extracellular Ca(2+). This study presents two new possible contributing factors, pH and Zn(2+). Plasma pH levels and micromolar concentrations of Zn(2+) inhibit WT Cx26 hemichannels. However, A40V KID mutant hemichannels show substantially reduced inhibition by these factors. Using excised patches, acidification was shown to be effective from either side of the membrane, suggesting a protonation site accessible to H(+) flux through the pore. Sensitivity to pH was not dependent on extracellular aminosulfonate pH buffers. Single channel recordings showed that acidification did not affect unitary conductance or block the hemichannel but rather promoted gating to the closed state with transitions characteristic of the intrinsic loop gating mechanism. Examination of two nearby KID mutants in the E1 domain, G45E and D50N, showed no changes in modulation by pH or Zn(2+). N-bromo-succinimide, but not thiol-specific reagents, attenuated both pH and Zn(2+) responses. Individually mutating each of the five His residues in WT Cx26 did not reveal a key His residue that conferred sensitivity to pH or Zn(2+). From these data and the crystal structure of Cx26 that suggests that Ala-40 contributes to an intrasubunit hydrophobic core, the principal effect of the A40V mutation is probably a perturbation in structure that affects loop gating, thereby affecting multiple factors that act to close Cx26 hemichannels via this gating mechanism.

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. Using electrophysiological measurements, we found that channels formed by Cx46 and Cx50, the two connexin isoforms expressed in the lens, were moderately cation-selective (P(Na)/P(Cl) ∼5 for Cx46 and ∼3 for Cx50). Single channel permeation of the larger GSH anion was low but detectable (P(Na)/P(GSH) ∼12 for Cx46 and ∼8 for Cx50), whereas permeation of divalent anion glutathione disulfide (GSSG) was undetectable. Measurement of GSH levels in the lenses from connexin knock-out (KO) mice indicated Cx46, and not Cx50, is necessary for transport of GSH to the core. Levels of GSH in the nucleus were markedly reduced in Cx46 KO, whereas they were unaffected by Cx50 KO. We also show that GSH delivery to the nucleus is not dependent on the lens microcirculation, which is believed to be responsible for extracellular transport of other nutrients to membrane transporters in the core. These results indicate that glutathione diffuses from cortical fiber cells to the nucleus via gap junction channels formed by Cx46. We present a model of GSH diffusion from outer cells to inner fiber cells through gap junctions.

Functional significance of thermosensitive transient receptor potential melastatin channel 8 (TRPM8) expression in immortalized human corneal endothelial cells.

Human corneal endothelial cells (HCEC) maintain appropriate tissue hydration and transparency by eliciting net ion transport coupled to fluid egress from the stroma into the anterior chamber. Such activity offsets tissue swelling caused by stromal imbibition of fluid. As corneal endothelial (HCE) transport function is modulated by temperature changes, we probed for thermosensitive transient receptor potential melastatin 8 (TRPM8) functional activity in immortalized human corneal endothelial cells (HCEC-12) and freshly isolated human corneal endothelial cells (HCEC) as a control. This channel is either activated upon lowering to 28 °C or by menthol, eucalyptol and icilin. RT-PCR and quantitative real-time PCR (qPCR) verified TRPM8 gene expression. Ca(2+) transients induced by either menthol (500 µmol/l), eucalyptol (3 mmol/l), or icilin (2-60 µmol/l) were identified using cell fluorescence imaging. The TRP channel blocker lanthanum III chloride (La(3+), 100 µmol/l) as well as the TRPM8 blockers BCTC (10 µmol/l) and capsazepine (CPZ, 10 µmol/l) suppressed icilin-induced Ca(2+) increases. In and outward currents induced by application of menthol (500 µmol/l) or icilin (50 µmol/l) were detected using the planar patch-clamp technique. A thermal transition from room temperature to ≈ 18 °C led to Ca(2+) increases that were inhibited by a TRPM8 blocker BCTC (10 µmol/l). Other thermosensitive TRP pathways whose heterogeneous Ca(2+) response patterns are suggestive of other Ca(2+) handling pathways were also detected upon strong cooling (≈10 °C). Taken together, functional TRPM8 expression in HCEC-12 and freshly dissociated HCEC suggests that HCE function can adapt to thermal variations through activation of this channel subtype.

CyclinD2-mediated regulation of neurogenic output from the retinal ciliary margin is perturbed in albinism.

In albinism, aberrations in the ipsi-/contralateral retinal ganglion cell (RGC) ratio compromise the functional integrity of the binocular circuit. Here, we focus on the mouse ciliary margin zone (CMZ), a neurogenic niche at the embryonic peripheral retina, to investigate developmental processes regulating RGC neurogenesis and identity acquisition. We found that the mouse ventral CMZ generates predominantly ipsilaterally projecting RGCs, but this output is altered in the albino visual system because of CyclinD2 downregulation and disturbed timing of the cell cycle. Consequently, albino as well as CyclinD2-deficient pigmented mice exhibit diminished ipsilateral retinogeniculate projection and poor depth perception. In albino mice, pharmacological stimulation of calcium channels, known to upregulate CyclinD2 in other cell types, augmented CyclinD2-dependent neurogenesis of ipsilateral RGCs and improved stereopsis. Together, these results implicate CMZ neurogenesis and its regulators as critical for the formation and function of the mammalian binocular circuit.

Retinal Ganglion Cell Axon Wiring Establishing the Binocular Circuit.

Binocular vision depends on retinal ganglion cell (RGC) axon projection either to the same side or to the opposite side of the brain. In this article, we review the molecular mechanisms for decussation of RGC axons, with a focus on axon guidance signaling at the optic chiasm and ipsi- and contralateral axon organization in the optic tract prior to and during targeting. The spatial and temporal features of RGC neurogenesis that give rise to ipsilateral and contralateral identity are described. The albino visual system is highlighted as an apt comparative model for understanding RGC decussation, as albinos have a reduced ipsilateral projection and altered RGC neurogenesis associated with perturbed melanogenesis in the retinal pigment epithelium. Understanding the steps for RGC specification into ipsi- and contralateral subtypes will facilitate differentiation of stem cells into RGCs with proper navigational abilities for effective axon regeneration and correct targeting of higher-order visual centers.

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. In HeLa cells, all three mutations also localized to regions of cell-cell contact and displayed a punctate staining pattern. In addition, we show that Cx43-G8V, Cx43-A44V and Cx43-E227D significantly increase membrane current flow through formation of active hemichannels, a novel activity that was not displayed by wild-type Cx43. The increased membrane current was inhibited by either 2 mM calcium, or 5 µM gadolinium, mediated by hemichannels with a unitary conductance of ~250 pS, and was not due to elevated mutant protein expression. The three Cx43 mutations all showed the same gain of function activity, suggesting that augmented hemichannel activity could play a role in skin-limited diseases caused by human Cx43 mutations.

Syndromic deafness mutations at Asn 14 differentially alter the open stability of Cx26 hemichannels.

Connexin 26 (Cx26) is a transmembrane protein that forms hexameric hemichannels that can function when unopposed or dock to form intercellular gap junction channels. Aberrantly functioning unopposed hemichannels are a common feature of syndromic deafness associated with mutations in Cx26. In this study, we examine two different mutations at the same position in the N-terminal domain of Cx26, N14K and N14Y, which have been reported to produce different phenotypes in patients. We find that both N14K and N14Y, when expressed alone or together with wild-type (WT) Cx26, result in functional hemichannels with widely disparate functional properties. N14K currents are robust, whereas N14Y currents are small. The two mutants also exhibit opposite shifts in voltage-dependent loop gating, such that activation of N14K and N14Y is shifted in the hyperpolarizing and depolarizing directions, respectively. Deactivation kinetics suggests that N14K stabilizes and N14Y destabilizes the open state. Single N14K hemichannel recordings in low extracellular Ca(2+) show no evidence of stable closing transitions associated with loop gating, and N14K hemichannels are insensitive to pH. Together, these properties cause N14K hemichannels to be particularly refractory to closing. Although we find that the unitary conductance of N14K is indistinguishable from WT Cx26, mutagenesis and substituted cysteine accessibility studies suggest that the N14 residue is exposed to the pore and that the differential properties of N14K and N14Y hemichannels likely result from altered electrostatic interactions between the N terminus and the cytoplasmic extension of TM2 in the adjacent subunit. The combined effects that we observe on loop gating and pH regulation may explain the unusual buccal cutaneous manifestations in patients carrying the N14K mutation. Our work also provides new considerations regarding the underlying molecular mechanism of loop gating, which controls hemichannel opening in the plasma membrane.

Identification and Functional Assessment of Age-Dependent Truncations to Cx46 and Cx50 in the Human Lens.

PURPOSE: Many proteins in the lens undergo extensive posttranslational modifications (PTMs) with age, leading to alterations in their function. The extent to which lens gap junction proteins, Cx46 and Cx50, accumulate PTMs with aging is not known. In this study, we identified truncations in Cx46 and Cx50 in the human lens using mass spectrometry. We also examined the effect of truncations on channel function using electrophysiological measurements. METHODS: Human lenses were dissected into cortex, outer nucleus, and nucleus regions, and fiber cell membranes were subjected to trypsin digestion. Tryptic peptides were analyzed by liquid chromatography (LC)-electrospray tandem mass spectrometry (ESI/MS/MS). Effects of truncations on channel conductance, permeability, and gating were assessed in transfected cells. RESULTS: Cleavage sites were identified in the C-terminus, the cytoplasmic loop, and the N-terminus of Cx46 and Cx50. Levels of C-terminal truncations, which were found at residues 238 to 251 in Cx46 and at residues 238 to 253 and 274 to 284 in Cx50, were similar in different lens regions. In contrast, levels of truncations in cytoplasmic loop and N-terminal domains of Cx46 and Cx50 increased dramatically from outer cortex to nucleus. Most of the C-terminally truncated proteins were functional, whereas truncations in the cytoplasmic loop did not result in the formation of functional channels. CONCLUSIONS: Accumulation of cytoplasmic loop and N-terminal truncations in the core might lead to decreases in coupling with age. This reduction is expected to lead to an increase in intracellular calcium and a decrease in levels of glutathione in the nucleus. These changes may ultimately lead to age-related nuclear cataracts.

Thyronamine induces TRPM8 channel activation in human conjunctival epithelial cells.

3-Iodothyronamine (T1AM), an endogenous thyroid hormone (TH) metabolite, induces numerous responses including a spontaneously reversible body temperature decline. As such an effect is associated in the eye with increases in basal tear flow and thermosensitive transient receptor potential melastatin 8 (TRPM8) channel activation, we determined in human conjunctival epithelial cells (IOBA-NHC) if T1AM also acts as a cooling agent to directly affect TRPM8 activation at a constant temperature. RT-PCR and quantitative real-time PCR (qPCR) along with immunocytochemistry probed for TRPM8 gene and protein expression whereas functional activity was evaluated by comparing the effects of T1AM with those of TRPM8 mediators on intracellular Ca(2+) ([Ca(2+)]i) and whole-cell currents. TRPM8 gene and protein expression was evident and icilin (20µM), a TRPM8 agonist, increased Ca(2+) influx as well as whole-cell currents whereas BCTC (10µM), a TRPM8 antagonist, suppressed these effects. Similarly, either temperature lowering below 23°C or T1AM (1µM) induced Ca(2+) transients that were blocked by this antagonist. TRPM8 activation by both 1µM T1AM and 20µM icilin prevented capsaicin (CAP) (20µM) from inducing increases in Ca(2+) influx through TRP vanilloid 1 (TRPV1) activation, whereas BCTC did not block this response. CAP (20µM) induced a 2.5-fold increase in IL-6 release whereas during exposure to 20µM capsazepine this rise was completely blocked. Similarly, T1AM (1µM) prevented this response. Taken together, T1AM like icilin is a cooling agent since they both directly elicit TRPM8 activation at a constant temperature. Moreover, there is an inverse association between changes in TRPM8 and TRPV1 activity since these cooling agents blocked both CAP-induced TRPV1 activation and downstream rises in IL-6 release.

BRI2 interacts with BACE1 and regulates its cellular levels by promoting its degradation and reducing its mRNA levels.

BRI2, a protein mutated in Familial British and Familial Danish Dementias, interacts with Amyloid Precursor Protein (APP) and reduces the levels of secreted APPß (sAPPß), which derives from APP cleavage by ß-secretase (BACE1). Exploring the mechanisms of this effect, we obtained data that BRI2 decreases the cellular levels of BACE1 thus reducing the ß-cleavage of APP. Deletion of N-terminal cytoplasmic or C-terminal extracellular sequences of BRI2 neither affected its interaction with BACE1 or APP (Fotinopoulou et al., 2005) nor the reduction in the levels of BACE1 and sAPPß. These results suggest that BRI2 may prevent access of BACE1 to APP and the BRI2/BACE1 interaction may mediate the reduction in BACE1 levels. In support, BRI2 expression induced lysosomal but not proteasomal degradation of BACE1. In parallel, BRI2 expression was also found to reduce BACE1 mRNA levels by 50%. This study adds novel information regarding the mechanism by which BRI2 affects APP processing and BACE1 levels.