Photoreceptor Ion Channels in Signaling and Disease.

Photoreceptors (PRs) in the neural retina convert photon capture into an electrical signal that is communicated across a chemical synapse to second-order neurons in the retina and on through the rest of the visual pathway. This information is decoded in the visual cortex to create images. The activity of PRs depends on the concerted action of several voltage-gated ion channels that will be discussed in this chapter.

Association of the Protein-Quality-Control Protein Ubiquilin-1 With Alzheimer's Disease Both in vitro and in vivo.

Alzheimer's disease (AD) belongs to a class of diseases characterized by progressive accumulation and aggregation of pathogenic proteins, particularly Aß proteins. Genetic analysis has identified UBQLN1 as an AD candidate gene. Ubiquilin-1 levels reduce with AD progression, suggesting a potential loss-of-function mechanism. The ubiquilin-1 protein is involved in protein quality control (PQC), which plays essential roles in cellular growth and normal cell function. Ubiquilin-1 regulates γ-secretase by increasing endoproteolysis of PS1, a key γ-secretase component. Presently, the effects of ubiquilin-1 on cellular physiology as well as Aß-related events require further investigation. Here, we investigated the effects of ubiquilin-1 on cellular growth and viability in association with APP (amyloid-ß protein precursor), APP processing-related ß-secretase (BACE1, BACE) and γ-secretase using cell and animal-based models. We showed that loss-of-function in Drosophila ubqn suppresses human APP and human BACE phenotypes in wing veins and altered cell number and tissue compartment size in the wing. Additionally, we performed cell-based studies and showed that silencing UBQLN1 reduced cell viability and increased caspase-3 activity. Overexpression of UBQLN1 significantly reduced Aß levels. Furthermore, pharmacological inhibition of γ-secretase increased ubiquilin-1 protein levels, suggesting a mechanism that regulates ubiquilin-1 levels which may associate with reduced Aß reduction by inhibiting γ-secretase. Collectively, our results support not only a loss-of-function mechanism of ubiquilin-1 in association with AD, but also support the significance of targeting ubiquilin-1-mediated PQC as a potential therapeutic strategy for AD.

Differential impact of Kv8.2 loss on rod and cone signaling and degeneration.

Heteromeric Kv2.1/Kv8.2 channels are voltage-gated potassium channels localized to the photoreceptor inner segment. They carry IKx, which is largely responsible for setting the photoreceptor resting membrane potential. Mutations in Kv8.2 result in childhood-onset cone dystrophy with supernormal rod response (CDSRR). We generated a Kv8.2 knockout (KO) mouse and examined retinal signaling and photoreceptor degeneration to gain deeper insight into the complex phenotypes of this disease. Using electroretinograms, we show that there were delayed or reduced signaling from rods depending on the intensity of the light stimulus, consistent with reduced capacity for light-evoked changes in membrane potential. The delayed response was not seen ex vivo where extracellular potassium levels were controlled by the perfusion buffer, so we propose the in vivo alteration is influenced by genotype-associated ionic imbalance. We observed mild retinal degeneration. Signaling from cones was reduced but there was no loss of cone density. Loss of Kv8.2 altered responses to flickering light with responses attenuated at high frequencies and altered in shape at low frequencies. The Kv8.2 KO line on an all-cone retina background had reduced cone-driven ERG b wave amplitudes and underwent degeneration. Altogether, we provide insight into how a deficit in the dark current affects the health and function of photoreceptors.

Transducin Partners Outside the Phototransduction Pathway.

Transducin mediates signal transduction in a classical G protein-coupled receptor (GPCR) phototransduction cascade. Interactions of transducin with the receptor and the effector molecules had been extensively investigated and are currently defined at the atomic level. However, partners and functions of rod transducin α (Gαt 1) and ßγ (Gß1γ1) outside the visual pathway are not well-understood. In particular, light-induced redistribution of rod transducin from the outer segment to the inner segment and synaptic terminal (IS/ST) allows Gαt1 and/or Gß1γ1 to modulate synaptic transmission from rods to rod bipolar cells (RBCs). Protein-protein interactions underlying this modulation are largely unknown. We discuss known interactors of transducin in the rod IS/ST compartment and potential pathways leading to the synaptic effects of light-dispersed Gαt1 and Gß1γ1. Furthermore, we show that a prominent non-GPCR guanine nucleotide exchange factor (GEF) and a chaperone of Gα subunits, resistance to inhibitors of cholinesterase 8A (Ric-8A) protein, is expressed throughout the retina including photoreceptor cells. Recent structures of Ric-8A alone and in complexes with Gα subunits have illuminated the structural underpinnings of the Ric-8A activities. We generated a mouse model with conditional knockout of Ric-8A in rods in order to begin defining the functional roles of the protein in rod photoreceptors and the retina. Our analysis suggests that Ric-8A is not an obligate chaperone of Gαt1. Further research is needed to investigate probable roles of Ric-8A as a GEF, trafficking chaperone, or a mediator of the synaptic effects of Gαt1.

A Comparison of the Primary Sensory Neurons Used in Olfaction and Vision.

Vision, hearing, smell, taste, and touch are the tools used to perceive and navigate the world. They enable us to obtain essential resources such as food and highly desired resources such as mates. Thanks to the investments in biomedical research the molecular unpinning's of human sensation are rivaled only by our knowledge of sensation in the laboratory mouse. Humans rely heavily on vision whereas mice use smell as their dominant sense. Both modalities have many features in common, starting with signal detection by highly specialized primary sensory neurons-rod and cone photoreceptors (PR) for vision, and olfactory sensory neurons (OSN) for the smell. In this chapter, we provide an overview of how these two types of primary sensory neurons operate while highlighting the similarities and distinctions.

Analysis of 14-3-3 isoforms expressed in photoreceptors.

The 14-3-3 family of proteins has undergone considerable expansion in higher eukaryotes with humans and mice expressing seven isoforms (ß, ε, η, γ, θ, ζ, and σ) from seven distinct genes (YWHAB, YWAHE, YWHAH, YWHAG, YWHAQ, YWHAZ, and SFN). Growing evidence indicates that while highly conserved, these isoforms are not entirely functionally redundant as they exhibit unique tissue expression profiles, subcellular localization, and biochemical functions. A key limitation in our understanding of 14-3-3 biology lies in our limited knowledge of cell-type specific 14-3-3 expression. Here we provide a characterization of 14-3-3 expression in whole retina and isolated rod photoreceptors using reverse-transcriptase digital droplet PCR. We find that all 14-3-3 genes with the exception of SFN are expressed in mouse retina with YWHAQ and YWHAE being the most highly expressed. Rod photoreceptors are enriched in YWHAE (14-3-3 ε). Immunohistochemistry revealed that 14-3-3 ε and 14-3-3 ζ exhibit unique distributions in photoreceptors with 14-3-3 ε restricted to the inner segment and 14-3-3 ζ localized to the outer segment. Our data demonstrates that, in the retina, 14-3-3 isoforms likely serve specific functions as they exhibit unique expression levels and cell-type specificity. As such, future investigations into 14-3-3 function in rod photoreceptors should be centered on 14-3-3 ε and 14-3-3 ζ, depending on the subcellular region of question.

Probing Functional Changes in Exocyst Configuration with Monoclonal Antibodies.

Spatial regulation of exocytosis relies on the exocyst, a hetero-octameric protein complex that tethers vesicles to fusion sites at the plasma membrane. Nevertheless, our understanding of mechanisms regulating exocyst assembly/disassembly, localization, and function are incomplete. Here, we have exploited a panel of anti-Sec6 monoclonal antibodies (mAbs) to probe possible configurational changes accompanying transitions in exocyst function in epithelial MDCK cells. Sec6 is quantitatively associated with Sec8 in high molecular weight complexes, as shown by gel filtration and co-immunoprecipitation studies. We mapped epitopes recognized by more than 20 distinct mAbs to one of six Sec6 segments. Surprisingly, mAbs that bound epitopes in each segment labeled distinct subcellular structures. In general, antibodies to epitopes in N-terminal domains labeled Sec6 in either cytosolic or nuclear pools, whereas those that bound epitopes in C-terminal domains labeled membrane-associated Sec6. In this latter group, we identified antibodies that labeled distinct Sec6 populations at the apical junctional complex, desmosomes, endoplasmic reticulum and vimentin-type intermediate filaments. That each antibody was specific was verified by both Sec6 RNAi and competition with fusion proteins containing each domain. Comparison of non-polarized and polarized cells revealed that many Sec6 epitopes either redistribute or become concealed during epithelial polarization. Transitions in exocyst configurations may be regulated in part by the actions of Ral GTPases, because the exposure of Sec6 C-terminal domain epitopes at the plasma membrane is significantly reduced upon RalA RNAi. To determine whether spatio-temporal changes in epitope accessibility was correlated with differential stability of interactions between Sec6 and other exocyst subunits, we quantified relative amounts of each subunit that co-immunoprecipitated with Sec6 when antibodies to N-terminal or C-terminal epitopes were used. Antibodies to Sec6NT co-precipitated substantially more Sec5, -10, -15, Exo70 and -84 than did those to Sec6CT. In contrast, antibodies to Sec6CT co-precipitated more Sec3 and Sec8 than did those to Sec6NT. These results are consistent with a model in which exocyst activation during periods of rapid membrane expansion is accompanied by molecular rearrangements within the holocomplex or association with accessory proteins, which expose the Sec6 C-terminal domain when the complex is membrane-bound and conceal it when the complex is cytoplasmic.

Syntaxin 16 regulates lumen formation during epithelial morphogenesis.

The formation and maintenance of cell-cell junctions, both under physiological and pathological conditions, requires the targeting and trafficking of junctional proteins. Proteins of the syntaxin (Stx)-family localize to a variety of subcellular membranes and contribute to intracellular transport of cargo by regulating vesicle fusion events at these sites. Unlike plasma membrane localized Stxs, the roles of endosome- and Golgi-localized stx proteins in epithelial morphogenesis are less understood. Here we show that Stx16- an endosome- and Golgi-localized target-membrane soluble N-ethylmaleimide attachment protein receptor (t-SNARE) that plays a role in membrane trafficking between these compartments - is essential for lumen development. In cultured Madin Darby Canine Kidney (MDCK) cells, Stx16 was selectively upregulated as sparsely plated cells attained confluency. Stx16-depleted confluent monolayers consistently showed lower transepithelial resistance than control monolayers, and failed to maintain endogenous and ectopically expressed E-cadherin at the adherens junctions due to decreased recycling. We further found that whereas cysts formed by MDCK cells cultured in Matrigel have a single hollow lumen, those formed by stx16-depleted counterparts had multiple lumens, due to abnormal orientiation of the mitotic spindle. Finally, a similar role for stx16 function in vivo is indicated by our analysis of pronephric-duct development in zebrafish expressing the claudinB:lynGFP transgene; lack of stx16 function in this structure (in stx16-morphant embryos) led to the development of enlarged, torturous pronephric ducts with more than one lumen. Taken together, our in vitro and in vivo studies establish a role for Stx16 in maintaining the integrity of cell-cell junctions, and thereby in morphogenesis of the kidney epithelial lumen.

The myosin motor Myo1c is required for VEGFR2 delivery to the cell surface and for angiogenic signaling.

Vascular endothelial growth factor receptor-2 (VEGFR2) is a receptor tyrosine kinase that is expressed in endothelial cells and regulates angiogenic signal transduction under both physiological and pathological conditions. VEGFR2 turnover at the plasma membrane (PM) is regulated by its transport through endocytic and secretory transport pathways. Short-range cargo trafficking along actin filaments is commonly regulated by motor proteins of myosin superfamily. In the current study, performed in primary human endothelial cells, we demonstrate that unconventional myosin 1c (Myo1c; class I family member) regulates the localization of VEGFR2 at the PM. We further demonstrate that the recruitment of VEGFR2 to the PM and its colocalization with Myo1c and caveolin-1 occur in response to VEGF-A (VEGF) stimulation. In addition, VEGF-induced delivery of VEGFR2 to the cell surface requires Myo1c; surface VEGFR2 levels are reduced in the absence of Myo1c and, more importantly, are restored by the overexpression of wild-type but not mutant Myo1c. Subcellular density gradient fractionation revealed that partitioning of VEGFR2 into caveolin-1- and Myo1c-enriched membrane fractions is dependent on VEGF stimulation. Myo1c depletion resulted in increased VEGF-induced VEGFR2 transport to the lysosomes for degradation and was rescued by applying either brefeldin A, which blocks trafficking between the endoplasmic reticulum and the Golgi complex, or dynasore, an inhibitor of dynamin-mediated endocytosis. Myo1c depletion also reduced VEGF-induced VEGFR2 phosphorylation at Y1175 and phosphorylation-dependent activation of ERK1/2 and c-Src kinase, leading to reduced cell proliferation and cell migration. This is the first report demonstrating that Myo1c is an important mediator of VEGF-induced VEGFR2 delivery to the cell surface and plays a role in angiogenic signaling.

Secretion of soluble vascular endothelial growth factor receptor 1 (sVEGFR1/sFlt1) requires Arf1, Arf6, and Rab11 GTPases.

The soluble form of vascular endothelial growth factor receptor 1 (sVEGFR-1/sFlt1) is generated by alternative splicing of the FLT1 gene. Secretion of sFlt1 from endothelial cells plays an important role in blood vessel sprouting and morphogenesis. However, excess sFlt1 secretion is associated with diseases such as preeclampsia and chronic kidney disease. To date, the secretory transport process involved in the secretion of sFlt1 is poorly understood. In the present study, we investigated the itinerary of sFlt1 trafficking along the secretory pathway. To understand the timecourse of sFlt1 secretion, endothelial cells stably expressing sFlt1 were metabolically radiolabeled with [(35)S]-methionine and cysteine. Our results indicate that after initial synthesis the levels of secreted [(35)S]-sFlt1 in the extracellular medium peaks at 8 hours. Treatment with brefeldin A (BFA), a drug which blocks trafficking between the endoplasmic reticulum (ER) and the Golgi complex, inhibited extracellular release of sFlt1 suggesting that ER to Golgi and intra-Golgi trafficking of sFlt1 are essential for its secretion. Furthermore, we show that ectopic expression of dominant-negative mutant forms of Arf1, Arf6, and Rab11 as well as siRNA-mediated knockdown of these GTPases block secretion of sFlt1 during normoxic and hypoxic conditions suggesting role for these small GTPases. This work is the first to report role of regulatory proteins involved in sFlt1 trafficking along the secretory pathway and may provide insights and new molecular targets for the modulation of sFlt-1 release during physiological and pathological conditions.