Notch signaling regulates Akap12 expression and primary cilia length during renal tubule morphogenesis.

Alagille syndrome patients present with loss of function mutations in either JAG1 or NOTCH2. About 40%-50% of patients have kidney abnormalities, and frequently display multicystic, dysplastic kidneys. Additionally, gain-of-function mutations in NOTCH2 are associated with cystic kidneys in Hajdu-Cheney syndrome patients. How perturbations in Notch signaling cause renal tubular cysts remains unclear. Here, we have determined that reduced Notch signaling mediated transcription by ectopic expression of dominant-negative mastermind-like (dnMaml) peptide in the nephrogenic epithelia from after the s-shaped body formation and in the developing collecting ducts results in proximal tubular and collecting duct cysts, respectively. An acute inhibition of Notch signaling for two days during kidney development is sufficient to disrupt tubule formation, and significantly increases Akap12 expression. Ectopic expression of Akap12 in renal epithelia results in abnormally long primary cilia similar to that observed in Notch-signaling-deficient epithelia. Both loss of Notch signaling and elevated Akap12 expression disrupt the ability of renal epithelial cells to form spherical structures with a single lumen when grown embedded in matrix. Interestingly, Akap12 can inhibit Notch signaling mediated transcription, which likely explains how both loss of Notch signaling and ectopic expression of Akap12 result in similar renal epithelial abnormalities. We conclude that Notch signaling regulates Akap12 expression while also ensuring normal primary cilia length and renal epithelial morphogenesis, and suggest that one aspect of diseases associated with defective Notch signaling, such as Alagille syndrome, maybe mechanistically related to ciliopathies.

Simultaneous Release of Multiple Vesicles from Rods Involves Synaptic Ribbons and Syntaxin 3B.

First proposed as a specialized mode of release at sensory neurons possessing ribbon synapses, multivesicular release has since been described throughout the central nervous system. Many aspects of multivesicular release remain poorly understood. We explored mechanisms underlying simultaneous multivesicular release at ribbon synapses in salamander retinal rod photoreceptors. We assessed spontaneous release presynaptically by recording glutamate transporter anion currents (IA(glu)) in rods. Spontaneous IA(glu) events were correlated in amplitude and kinetics with simultaneously measured miniature excitatory postsynaptic currents in horizontal cells. Both measures indicated that a significant fraction of events is multiquantal, with an analysis of IA(glu) revealing that multivesicular release constitutes ∼30% of spontaneous release events. IA(glu) charge transfer increased linearly with event amplitude showing that larger events involve greater glutamate release. The kinetics of large and small IA(glu) events were identical as were rise times of large and small miniature excitatory postsynaptic currents, indicating that the release of multiple vesicles during large events is highly synchronized. Effects of exogenous Ca2+ buffers suggested that multiquantal, but not uniquantal, release occurs preferentially near Ca2+ channels clustered beneath synaptic ribbons. Photoinactivation of ribbons reduced the frequency of spontaneous multiquantal events without affecting uniquantal release frequency, showing that spontaneous multiquantal release requires functional ribbons. Although both occur at ribbon-style active zones, the absence of cross-depletion indicates that evoked and spontaneous multiquantal release from ribbons involve different vesicle pools. Introducing an inhibitory peptide into rods to interfere with the SNARE protein, syntaxin 3B, selectively reduced multiquantal event frequency. These results support the hypothesis that simultaneous multiquantal release from rods arises from homotypic fusion among neighboring vesicles on ribbons and involves syntaxin 3B.

Elf5 is a principal cell lineage specific transcription factor in the kidney that contributes to Aqp2 and Avpr2 gene expression.

The mammalian kidney collecting ducts are critical for water, electrolyte and acid-base homeostasis and develop as a branched network of tubular structures composed of principal cells intermingled with intercalated cells. The intermingled nature of the different collecting duct cell types has made it challenging to identify unique and critical factors that mark and/or regulate the development of the different collecting duct cell lineages. Here we report that the canonical Notch signaling pathway components, RBPJ and Presinilin1 and 2, are involved in patterning the mouse collecting duct cell fates by maintaining a balance between principal cell and intercalated cell fates. The relatively reduced number of principal cells in Notch-signaling-deficient kidneys offered a unique genetic leverage to identify critical principal cell-enriched factors by transcriptional profiling. Elf5, which codes for an ETS transcription factor, is one such gene that is down-regulated in kidneys with Notch-signaling-deficient collecting ducts. Additionally, Elf5 is among the earliest genes up regulated by ectopic expression of activated Notch1 in the developing collecting ducts. In the kidney, Elf5 is first expressed early within developing collecting ducts and remains on in mature principal cells. Lineage tracing of Elf5-expressing cells revealed that they are committed to the principal cell lineage by as early as E16.5. Over-expression of ETS Class IIa transcription factors, including Elf5, Elf3 and Ehf, increase the transcriptional activity of the proximal promoters of Aqp2 and Avpr2 in cultured ureteric duct cell lines. Conditional inactivation of Elf5 in the developing collecting ducts results in a small but significant reduction in the expression levels of Aqp2 and Avpr2 genes. We have identified Elf5 as an early maker of the principal cell lineage that contributes to the expression of principal cell specific genes.

Conjunctival Lesions Secondary to Systemic Varicella Zoster Virus Infection.

Purpose: To report and present images of a case in which discrete conjunctival lesions developed in the setting of primary varicella zoster virus infection (ie, chickenpox). Methods: Case report and literature review. Results: This report describes a young, unvaccinated male who developed an acutely painful, red eye in the setting of disseminated primary varicella zoster infection. The cutaneous rash was widespread and included lesions on both eyelids. The patient was found to have multiple discrete de-epithelialized lesions involving the palpebral and bulbar conjunctiva. Throughout the disease course, good visual function was maintained and there was no evidence of intraocular involvement. The ocular surface lesions resolved without sequelae after 1 week of treatment with topical antibiotic ointment. Conclusions: Primary varicella zoster infection is an increasingly rare phenomenon in the setting of widespread vaccination. However, unvaccinated or undervaccinated individuals and other at-risk populations remain susceptible to developing severe infections. This case of chickenpox involved discrete conjunctival lesions that resolved without sequelae after conservative treatment with topical antibiotic ointment. While serious ophthalmic complications are uncommon in primary varicella infection, clinicians should be aware of the potential for ocular morbidity in this increasingly rare condition.

Diffuse Lamellar Keratitis in a Patient Undergoing Collagen Corneal Cross-Linking 18 Years After Laser In Situ Keratomileusis Surgery.

PURPOSE: To report a case of diffuse lamellar keratitis (DLK) after corneal collagen cross-linking in an eye with a remote history of laser in situ keratomileusis (LASIK) surgery. METHODS: This is a case report and literature review. RESULTS: This report describes the development of unilateral stage IV DLK in a patient who underwent bilateral corneal cross-linking for corneal ectasia 18 years after LASIK surgery. The patient was treated with high-dose topical steroids that were tapered over 1 month and multiple flap lifts. The ultimate best-corrected visual outcome was 20/60. CONCLUSIONS: DLK is a potential sight-threatening complication of refractive surgery that can occur at any time in the postoperative period, even years after the procedure. Undergoing a subsequent corneal procedure that may disrupt or promote inflammation within the surgical flap-stromal interface, such as corneal collagen cross-linking, is a recognized risk factor for the development of DLK. This case suggests that patients with any history of LASIK surgery undergoing corneal cross-linking or other lamellar corneal surgeries may benefit from closer follow-up (eg, daily) than patients with no history of LASIK.

Corrigendum: Synaptic Ribbon Active Zones in Cone Photoreceptors Operate Independently From One Another.

[This corrects the article DOI: 10.3389/fncel.2017.00198.].

Ca2+ sensor synaptotagmin-1 mediates exocytosis in mammalian photoreceptors.

To encode light-dependent changes in membrane potential, rod and cone photoreceptors utilize synaptic ribbons to sustain continuous exocytosis while making rapid, fine adjustments to release rate. Release kinetics are shaped by vesicle delivery down ribbons and by properties of exocytotic Ca2+ sensors. We tested the role for synaptotagmin-1 (Syt1) in photoreceptor exocytosis by using novel mouse lines in which Syt1 was conditionally removed from rods or cones. Photoreceptors lacking Syt1 exhibited marked reductions in exocytosis as measured by electroretinography and single-cell recordings. Syt1 mediated all evoked release in cones, whereas rods appeared capable of some slow Syt1-independent release. Spontaneous release frequency was unchanged in cones but increased in rods lacking Syt1. Loss of Syt1 did not alter synaptic anatomy or reduce Ca2+ currents. These results suggest that Syt1 mediates both phasic and tonic release at photoreceptor synapses, revealing unexpected flexibility in the ability of Syt1 to regulate Ca2+-dependent synaptic transmission.

Endocytosis sustains release at photoreceptor ribbon synapses by restoring fusion competence.

Endocytosis is an essential process at sites of synaptic release. Not only are synaptic vesicles recycled by endocytosis, but the removal of proteins and lipids by endocytosis is needed to restore release site function at active zones after vesicle fusion. Synaptic exocytosis from vertebrate photoreceptors involves synaptic ribbons that serve to cluster vesicles near the presynaptic membrane. In this study, we hypothesize that this clustering increases the likelihood that exocytosis at one ribbon release site may disrupt release at an adjacent site and therefore that endocytosis may be particularly important for restoring release site competence at photoreceptor ribbon synapses. To test this, we combined optical and electrophysiological techniques in salamander rods. Pharmacological inhibition of dynamin-dependent endocytosis rapidly inhibits release from synaptic ribbons and slows recovery of ribbon-mediated release from paired pulse synaptic depression. Inhibiting endocytosis impairs the ability of second-order horizontal cells to follow rod light responses at frequencies as low as 2 Hz. Inhibition of endocytosis also increases lateral membrane mobility of individual Ca2+ channels, showing that it changes release site structure. Visualization of single synaptic vesicles by total internal reflection fluorescence microscopy reveals that inhibition of endocytosis reduces the likelihood of fusion among vesicles docked near ribbons and increases the likelihood that they will retreat from the membrane without fusion. Vesicle advance toward the membrane is also reduced, but the number of membrane-associated vesicles is not. Endocytosis therefore appears to be more important for restoring later steps in vesicle fusion than for restoring docking. Unlike conventional synapses in which endocytic restoration of release sites is evident only at high frequencies, endocytosis is needed to maintain release from rod ribbon synapses even at modest frequencies.

Synaptic Ribbon Active Zones in Cone Photoreceptors Operate Independently from One Another.

Cone photoreceptors depolarize in darkness to release glutamate-laden synaptic vesicles. Essential to release is the synaptic ribbon, a structure that helps organize active zones by clustering vesicles near proteins that mediate exocytosis, including voltage-gated Ca2+ channels. Cone terminals have many ribbon-style active zones at which second-order neurons receive input. We asked whether there are functionally significant differences in local Ca2+ influx among ribbons in individual cones. We combined confocal Ca2+ imaging to measure Ca2+ influx at individual ribbons and patch clamp recordings to record whole-cell ICa in salamander cones. We found that the voltage for half-maximal activation (V50) of whole cell ICa in cones averaged -38.1 mV ± 3.05 mV (standard deviation [SD]), close to the cone membrane potential in darkness of ca. -40 mV. Ca2+ signals at individual ribbons varied in amplitude from one another and showed greater variability in V50 values than whole-cell ICa, suggesting that Ca2+ signals can differ significantly among ribbons within cones. After accounting for potential sources of technical variability in measurements of Ca2+ signals and for contributions from cone-to-cone differences in ICa, we found that the variability in V50 values for ribbon Ca2+ signals within individual cones showed a SD of 2.5 mV. Simulating local differences in Ca2+ channel activity at two ribbons by shifting the V50 value of ICa by ±2.5 mV (1 SD) about the mean suggests that when the membrane depolarizes to -40 mV, two ribbons could experience differences in Ca2+ influx of >45%. Further evidence that local Ca2+ changes at ribbons can be regulated independently was obtained in experiments showing that activation of inhibitory feedback from horizontal cells (HCs) to cones in paired recordings changed both amplitude and V50 of Ca2+ signals at individual ribbons. By varying the strength of synaptic output, differences in voltage dependence and amplitude of Ca2+ signals at individual ribbons shape the information transmitted from cones to downstream neurons in vision.