Catherine Morgans - US grants

DESCRIPTION (provided by applicant): Recently the gene causing incomplete congenital stationary night blindness (CSNB2) was identified and found to encode a retina-specific, voltage-activated calcium channel, alpha-1F. The disease is characterized by severely reduced nighttime (or rod-mediated) vision as well as abnormalities in daytime (or cone-mediated) vision. The electrophysiological and psychophysical phenotype of CSNB2 can be explained by a defect in synaptic transmission within the retina. We have found that the alpha-1F calcium channel is localized to rod photoreceptor and rod bipolar cell synaptic terminals in the rat and mouse retinas consistent with the night-blindness associated with CSNB2. Voltage-activated calcium channels are essential to the first two stages of visual processing in the retina, which occur at photoreceptor and bipolar cell ribbon synapses. At the ribbon synapse, calcium channels couple the graded electrical responses of the photoreceptor or bipolar cell to calcium-dependent, graded release of the excitatory neurotransmitter, glutamate. Our data suggest that the symptoms of CSNB2 are caused by a block of glutamate release at photoreceptor and bipolar cell ribbon synapses. A detailed characterization of the alpha -1F calcium channel is essential to understanding both the complex phenotype of CSNB2 and normal synaptic transmission in the retina. Yet the biophysical properties of alpha-1F calcium channels are not known. Nor is it known whether alpha-1F is present in cone photoreceptor and cone bipolar cell terminals, or whether other calcium channels mediate glutamate release in the cone pathway. The proposed experiments will address these questions. The Specific Aims are to 1. Determine in detail the distribution and functional properties of the alpha-1F calcium channel subunit. 2. Identify retinal proteins that interact with alpha-1F. 3. Identify other voltage-activated calcium channels at retinal ribbon synapses. We will use a multidisciplinary approach involving molecular, biochemical, immunohistochemical and electrophysiological techniques to achieve these aims. The results of these experiments will provide insight into the composition and functional organization of retinal ribbon synapses and into the perturbation of retinal function associated with CSNB2.

[unreadable] DESCRIPTION (provided by applicant): Complete X-linked congenital stationary night blindness (CSNB1) is a hereditary disease caused by a block in synaptic transmission in the retina between photoreceptors and ON-bipolar cells (ON-BPCs). Photoreceptors release the neurotransmitter, glutamate, in darkness, and reduce the rate of release in response to light. ON-BPCs respond to glutamate via the mGluR6 metabotropic glutamate receptor. Binding of glutamate by mGluR6 leads to the closure of a cation channel and hyperpolarization of the ON-BPC. The role of mGluR6 in transducing the action of glutamate in the ON-BPC dendrites is central to vertebrate vision, yet the steps in the mGluR6 signaling pathway are poorly understood, and the molecular identity of the ON-BPC cation channel is unknown. The human gene responsible for CSNB1, NYX, encodes a novel protein, nyctalopin, belonging to the family of leucine rich repeat (LRR) proteins. A mouse mutant, nob (no b-wave) has a similar phenotype to CSNB1 and a deletion in the nyx gene. Expression of mGluR6 appears normal in the nob mice, however their ON-BPCs fail to respond to exogenously applied glutamate, implicating nyctalopin in the mGluR6 signaling pathway. Although essential for the ON-BPC light response, the cellular function of nyctalopin is not known. Related LRR proteins in the nervous system have been implicated in neurite outgrowth and synapse formation, where they bind other proteins through their LRR domains including soluble ligands, receptors, and G proteins. We propose that nyctalopin is found at the photoreceptor to ON-BPC synapse where it interacts with components of the mGluR6 signaling pathway. To test this hypothesis, we will determine the localization of nyctalopin in the retina by immunofluorescence microscopy and immuno-electron microscopy using antibodies directed against a unique nyctalopin peptide. We will identify proteins interacting with nyctalopin by immunoprecipitation and yeast two-hybrid screening. These results will provide insight into the role of nyctalopin in synaptic transmission between photoreceptors and ON-BPCs in the retina, and may lead to the identification of other components of the ON-BPC pathway. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): In the retina, visual information is quickly segregated into pathways that respond to increases and decreases in light intensity. At the first retinal synapse, the tonic release of glutamate from photoreceptor terminals maintains a high synaptic concentration in darkness that rapidly decreases in response to light. Two types of postsynaptic cells, the ON- and OFF-bipolar cells (BPCs), respond with opposite polarity to glutamate released by photoreceptors, thus establishing the opposing visual pathways that are maintained throughout the rest of the visual system. The basis of signaling in OFF-BPCs, which relies on the activation of ionotropic glutamate receptors, is well understood;the signaling pathway that generates the light response in ON-BPCs, however, is more complex, and the molecular mechanisms remain to be elucidated. The ON-BPC signaling pathway originates with a unique metabotropic glutamate receptor, mGluR6, which is found exclusively on the dendrites of ON-BPCs. mGluR6 acts via a G-protein, Go, to regulate the activity of an unidentified cation channel such that the light-induced decrease in glutamate opens the channel and depolarizes the cell. In many ways, this sequence of events resembles the well-studied signal transduction pathway of photoreceptor outer segments, in which photoexcitation of rhodopsin is coupled via the G-protein, transducin, to the closure of a cGMP-gated cation channel. In the outer segment, the kinetics of the light response is largely determined by the lifetime of activated transducin. Deactivation of transducin occurs upon hydrolysis of GTP by the transducin alpha subunit, and this reaction is accelerated by interaction with the G[unreadable]5-RGS9-R9AP complex. Mutations in the gene encoding any one of these three proteins severely impair vision by slowing recovery after light flashes. We have identified two similar complexes, G[unreadable]5-RGS7 and G[unreadable]5-RGS11, in ON-BPC dendrites suggesting a similar mechanism of deactivation of the ON-BPC signal transduction pathway. We hypothesize that the RGS-G[unreadable]5 complexes are critical components of the mGluR6 signal transduction pathway in ON-BPC dendrites. Using a combination of biochemical, immunohistochemical, and electrophysiological approaches, we will test this hypothesis by answering the following questions: 1. How do RGS-G[unreadable]5 complexes shape the response of ON-BPCs to light? 2. How are these RGS complexes anchored in the ON-BPC dendrites and how does this affect their function? 3. What other proteins in the mGluR6 pathway interact with G[unreadable]5-RGS7 and G[unreadable]5-RGS11? The data from this study will contribute to the elucidation of the signaling pathway in the ON-bipolar cell, a fundamental, yet poorly understood, step in visual processing. PUBLIC HEALTH RELEVANCE: The proposed research into the intracellular biochemical pathways generating the light response of retinal ON-bipolar cells is of relevance to the development of cures for blindness, including the design of visual prosthetics and gene therapy. Moreover, because G protein-coupled receptors and pathways are the target of the majority of pharmaceutical drugs, this research will have broad relevance to the development of therapeutic interventions for numerous neurological and cardiovascular diseases.

DESCRIPTION (provided by applicant): Melanoma Associated Retinopathy (MAR) is a paraneoplastic visual syndrome associated with cutaneous malignant melanoma in which patients typically experience a sudden decrease in night vision and sensations of shimmering lights. Electroretinogram (ERG) recordings from MAR patients are characterized by a negative waveform in which the a-wave, arising from photoreceptor transduction, is normal, but the b-wave, arising primarily from ON-bipolar cell activation, is absent or reduced. Significantly, serum from MAR patients has been shown to label retinal bipolar cells. The proposed research tests the hypothesis that autoantibodies in MAR patient serum are generated against proteins expressed in metastatic malignant melanocytes, but then target a protein complex functional in bipolar cells. The aims of the research are to identify the retinal antigens in MAR, to elucidate the role of these proteins in normal bipolar cell physiology, and determine how these functions are perturbed in MAR. The aims will be addressed with a multidisciplinary approach combining immunohistochemistry, cell biology, biochemistry, and electrophysiology. The results will generate new insights into cellular processes fundamental to vision and will contribute to our understanding of the link between cancer and autoimmune retinopathy.

PROJECT SUMMARY - BIO-IMAGING AND CONFOCAL MICROSCOPY The Bio-Imaging & Confocal Microscopy Core at OHSU will provide instrumentation and support for fluorescence and confocal microscopy as well as spectral domain optical coherence tomography (SDOCT) to enhance and facilitate vision research at OHSU. The major instrumentation and expertise are already in place at the Casey Eye Institute (CEI). Past P30 support of confocal and fluorescence imaging systems at the CEI has been extremely successful, with data from the supported instruments featuring in 50 publications during the previous 5-year funding period. Changes in the next funding period include the addition of small animal SDOCT instrumentation to the Core. The Bioptigen SDOCT device makes possible repeated, noninvasive, in vivo imaging of the retina and eye and will substantially enhance ongoing and planned research projects at CEI aimed at improving the treatment of visual diseases. Dr. Catherine Morgans, an expert in confocal and fluorescence microscopy, will continue to serve as Director of the Bio-Imaging Core, and Dr. Mark Pennessi, expert at rodent SDOCT, along with Dr. David Huang, co-inventor of OCT technology, will provide guidance and training to new SDOCT users. Training opportunities on all instruments will be available to principal investigators, postdoctoral fellows, and graduate students.

Project Summary The Oregon Health & Science University (OHSU) Neuroscience Imaging Center will provide expertise in designing, conducting, and analyzing experiments on state-of-the-art instrumentation for light and electron microscopy. The Neuroscience Imaging Center has two scientific Cores: Core 1 - Analytical Fluorescence Imaging and Core 2 - Ultrastructure and Single Particle Microscopy. The instruments for these Cores are in place and the technical capacities have been expanded tremendously over the past six years to include not only confocal microscopy and transmission electron microscopy, but also in vivo two-photon microscopy, super-resolution microscopy, EM tomography and single particle analyses for determining molecular structures. All instruments are located in university-wide service centers that are supported by the office of the Vice President for Research. The Neuroscience Imaging Center serves NINDS-funded OHSU researchers, as well as other NIH-funded investigators engaged in neuroscience research. We currently have 21 NINDS- funded investigators who have submitted qualifying projects for the current proposal and have microscopy and imaging needs for their current research. Other OHSU neuroscientists also receive support from Core staff. User fees on shared instruments are partially supported by the P30 grant, with greater support for NINDS than non-NINDS investigators; user fee support is regularly reviewed by the Steering Committee. Microscopy core facilities are located on the Marquam Hill campus and at the adjacent Waterfront Campus (see Resources), and all P30-supported investigators will have open access to all of these resources. The OHSU Neuroscience Imaging Center grant provides salary support for Core staff, who are local, service-oriented technology experts. The Neuroscience Imaging Center Director Dr. Sue Aicher and Associate Director Dr. Catherine Morgans have extensive scientific and technical expertise and will provide front-end consultation with investigators, helping to direct each investigator to the appropriate resources to address their research goals. The Steering Committee provides input to the Center Director with regard to Core strategic and operational issues, as well as project prioritization and Core access plans. We provide different levels of support based on the needs of the investigators. Investigators with extensive imaging expertise primarily require access to state- of-the-art equipment and occasional consultation with staff. Other investigators need more in-depth support, and full service support is often provided for electron microscopy studies. The P30 staff will provide expert support at the appropriate level to meet the needs of our participating investigators. The Neuroscience Imaging Center has catalyzed interactions among NINDS researchers as well as other neuroscientist at OHSU, provided synergy for investments in advanced microscopy, and significantly enhanced scientific progress for our neuroscience community.

Project Summary/Abstract: A group of National Eye Institute (NEI)-funded investigators at the Casey Eye Institute of Oregon Health & Science University requests funds to purchase a Leica TCS SP8 X confocal microscope to be placed in their P30-funded Bio-imaging and Confocal Microscopy Core. The TCS SP8 X will replace an aging Olympus FV1000, which has been in place since early 2005. The high resolution, sensitivity and fast scan rates of the new instrument will increase both the quality and productivity of vision-related research at OHSU. Participating projects include studies on the causes and treatment of major diseases affecting vision, including glaucoma, uveitis, retinal degenerative diseases, and autoimmune retinopathies, as well as research on fundamental mechanisms underlying normal visual function. After careful market evaluation, the TCS SP8 X was selected as the most versatile, high-performing system capable of meeting the current and projected imaging needs of the participating users. The instrument will be maximally utilized by placement in the Bio-Imaging and Confocal Microscopy Core, where it will be supported by the expertise and organization of a well established, NEI P30-funded Center. Casey Eye Institute commits to ensuring long-term, efficient use of the instrument by covering costs of the service contract and personnel salary via the P30 grant and as needed. Instrument time not used by the NEI investigators on this application will be available to other scientists at Oregon Health & Science University, thereby extending the overall benefits. The introduction of this instrument will fulfill the growing needs of the vision research community at OHSU to incorporate state-of-the-art confocal microscopy into their research. This will speed the progress of discovery to improve the detection and treatment of human diseases affecting vision.

Project Summary Some cutaneous malignant melanoma (CMM) patients experience a sudden and rapid decline in their night vision often accompanied by photophobia and a sensation of shimmering light. These symptoms are a hallmark of a paraneoplastic autoimmune syndrome known as melanoma-associated retinopathy (MAR), which is clinically diagnosed by a reduced b-wave on the electroretinogram. We and others have identified the TRPM1 cation channel as the autoantigen. TRPM1 channels are expressed in melanocytes and retinal ON- bipolar cells, thus autoantibodies against TRPM1 block ON bipolar cell responses. A tumor suppressor microRNA, miR-211, is encoded within the 6th intron of TRPM1 and co-transcribed with TRPM1. Full-length TRPM1 and miR-211 are down regulated in metastatic disease, yet this is when TRPM1 autoantibodies are typically detected. We propose that the autoantibodies are generated against truncated, antigenic TRPM1 polypeptides encoded by abnormal TRPM1 mRNA splice variants, associated with reduced expression of miR-211.. The overall rationale of the proposed studies is that the occurrence of TRPM1 autoantibodies is more widespread in CMM patients than suggested by the incidence of clinically diagnosed MAR, and that the increased use of targeted and immuno therapies may heighten the risk of MAR. The proposed project aims to determine the incidence of TRPM1 autoantibodies and sub-clinical MAR among CMM patients and whether it varies according to treatment. Further, we aim to identify which TRPM1 mRNA splice variants give rise to immunoreactive TRPM1 polypeptides and test our hypothesis that these polypeptides are present in CMM specimens from patients with TRPM1 autoantibodies and sub-clinical MAR, and are associated with a down- regulation of miR-211. Thus, we will generate new insights into the cellular mechanisms underlying MAR, which may be further relevant to paraneoplastic autoimmune diseases in general. Potential applications of this research include the development of a prognostic/diagnostic test that can be used in the clinic for assessing CMM patients' risk of MAR and tumor metastasis.

The retina is exposed to light intensities that vary over nine orders of magnitude, from a cloudy night in a forest to a sunny day on a snowy mountainside, and to images of varying contrast and frequency. To optimize vision over this entire range, the response properties of the retina change as a function of the stimuli at both the cellular and network level, a process termed adaptation. The long-term goal of the proposed research is to explain the molecular basis for regulation of the light response in retinal ON-bipolar cells. These cells mediate the transmission of light responses between photoreceptors and ganglion cells and are key sites of adaptation. Rod bipolar cells receive light-driven synaptic input from rod photoreceptors and drive retinal output via synapses onto AII amacrine cells. While dark-adapted rod bipolar cells can transmit single photon responses in starlight, they are also able to transmit contrast changes in moderate background light. The mechanisms which optimize rod bipolar cell function under different lighting conditions remain unknown. Our recent work suggests that a novel mGlu5-based pathway operating in parallel to the primary light-response pathway may modulate the ON-bipolar cell responses. Further, we have identified a potassium channel, Kv11.1, that appears to regulate dark adaptation, and may be regulated by PKC?, which is abundantly expressed in rod bipolar cells. In the dark, photoreceptors release glutamate onto dendrites of ON-bipolar cells, and decrease glutamate release in response to light stimuli. The light response of ON-bipolar cells is mediated by a unique, sign- inverting pathway initiated by mGlu6, a G protein-coupled receptor in the ON-bipolar cell dendrites. In the dark, tonic activation of the mGlu6 pathway maintains the TRPM1 cation channel in a closed state. In response to light stimuli, mGlu6 is inactivated, allowing TRPM1 channels to open and depolarize the cell. The mGlu6- TRPM1 pathway is conserved in all vertebrates, and mutations in mGlu6 and TRPM1 cause congenital stationary night blindness (CSNB) in humans and mouse models. Despite its central importance in vision, the molecular mechanisms by which the primary excitatory pathway is modulated under different conditions remain unknown. Based on analogy with other systems, and our Preliminary Studies, we hypothesize that mGlu5 receptors, Kv11.1 channels and PKC? modulate the output of the mGlu6-TRPM1 pathway.

SUMMARY Electrical synapses, also known as gap junctions, occur frequently in all nervous systems, including the human brain. They are composed of connexins, arranged to form intercellular channels between adjacent, coupled cells. Connexin36 (Cx36) is the predominant connexin in the CNS. In many brain and retinal circuits, gap junctions provide direct and specific connections between cells. In addition, electrical synapses mediate network properties such as signal averaging, noise reduction and synchronization. However, because of their small size, gap junctions are not visible in large-scale serial EM data sets. For these reasons, gap junctions tend to be under-reported or simply ignored. The objective of this proposal is to develop a combined approach to image gap junction connectivity in EM datasets and, in addition, to estimate the size, strength, and plasticity of gap junctions. We will study regions of the retina that contain gap junctions of dramatically different sizes and shapes, to allow us to correlate structure and function. Aim 1 will use high-resolution confocal microscopy to determine connexon number at large and small gap junctions. Analyses will determine the number of connexons per gap junction. These methods will provide a general-purpose tool to determine the size of gap junctions for use in all brain regions. Aim 2 will use 3D-EM imaging to allow unambiguous identification of gap junctions in FIB-SEM images, which will follow with first-ever immunogold quantification of a membrane-bound protein in 3D-EM structures. These studies will allow high-resolution quantification of gap junctions and proteins in identified neurons. Aim 3 will use electrophysiological measures to determine coupling conductance and then develop models to calculate the maximal potential coupling conductance from the morphological data by multiplying the number of channels/gap junction [Specific Aim 1] times the connectivity (the number of gap junctions between coupled cells) [Specific Aim 2], times the unitary conductance of Cx36. Using paired recordings, we will obtain direct physiological measures of the junctional conductance between coupled cells. Then, by comparison with the potential maximum calculated from the morphological data, we can calculate the open channel probability and place realistic limits on the operating range. These are the fundamental properties required to understand the function of gap junctions in neuronal microcircuits. This program is an exact match for one of the listed areas, ?Tools to identify gap junctions and characterize electrical synapses? in the Funding Opportunity Announcement, RFA-MH-20-135.