Robert M. Duvoisin, PhD - US grants

DESCRIPTION: (Applicant's Abstract) The goal of the laboratory is to better understand the molecular mechanisms of neural processing in the retina. It is known that the segregation of light stimuli into ON and OFF pathways is done at the level of the bipolar cells. Since it is thought that photoreceptor cells release glutamate as a neurotransmitter, the functional difference between ON and OFF bipolar cells is proposed to be the result of their expressing distinct glutamate receptors. Specifically this proposal aims to explore the relationship between the expression of different glutamate receptors and the diversity of bipolar cells in the mouse retina. 1.Glutmate receptors in OFF bipolar cells. The diversity and distribution of glutamate receptor subunits will be examined in OFF bipolar cells by in situ hybridization and immunonohistochemical analyses of horizontal and vertical retinal sections and dissociated retinal cells. Glutamate receptor subunit expression in electrophysiologically identified individual OFF bipolar cells will be determined using the PCR technique. 2.Glutamate receptors in ON bipolar cells. The APB-type glutamate receptor, which is thought to mediate the sign inversion in ON bipolar cells, will be cloned. Antibodies will be raised against fragments of the APB receptor expressed in bacteria. The diversity and distribution of glutamate receptors will be examined in ON bipolar cells by in situ hybridization and immunohistochemical analyses of horizontal and vertical retinal sections and dissociated retinal cells. Glutamate receptor expression in electrophysiologically identified individual ON bipolar cells will be analyzed using the PCR technique. 3.Glutamate receptors during development. Studies have shown an early exrpession of neurotransmitter receptors in the developing brain, suggesting some role in the establishment of neural circuitry. The regulation of glutamate receptor expression in relation to the differentiation of bipolar cells will be analyzed using in situ hybridization and immunohistochemistry. The research proposed here will provide the basis with which to analyze the expression of glutamte receptors in mice with retinal degenerations and to assess the effects of receptor alterations during development and in the adult retina using transgenic mice.

The nucleus and organelles of lens fiber cells are degraded during cell differentiation. Because light would be scattered by these membranous particles, this process is necessary for clear vision. Very little is known about the biochemical and cellular mechanisms of this degradation and their regulation. A similar phenomenon occurs in reticulocytes, precursors of red blood cells. Following nucleus expulsion, organelles, including mitochondria and endoplasmic reticulum, are degraded. It has been proposed for a number of years that this process involves lipoxygenase, an enzyme that dioxygenases arachidonic acid and other polyenoic fatty acids. Several mechanisms have been suggested, all based on lipoxygenase modifying organelle membrane lipids or membrane-associated proteins. We have accumulated data that support a totally new mechanism: we propose that the soluble enzyme lipoxygenase assembles into a multimeric structure that forms pores in the membranes of organelles. Such pores would allow the cytoplasmic protein degradation machinery to gain access to the lumenal compartment and initiate the degradation of the organelle. We also found that lipoxygenase is expressed in the lens, most strongly in the peripheral fiber cells where nucleus and organelle degradation occur. We hypothesize that organelle degradation in the lens uses a similar mechanism as in reticulocytes. This proposal will test this hypothesis by identifying the lipoxygenase isozyme expressed in lens, analyzing its regulation of expression, gaining a better understanding of how lipoxygenase permeates membranes, and studying the expression of lipoxygenase in animal model and human cataractous lenses.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] [unreadable] This application for a new, institutional training grant requests funds for 4 positions (2 in the initial year), for postdoctoral fellows. Fellows will participate in the recently-developed Training Program in the Neurological Sciences at the Neurological Sciences Institute (NSI), a multi-disciplinary neuroscience research unit at Oregon Health & Science University (OHSU). NSI faculty is a diverse group with primary training in biological sciences, physics, engineering, and physical therapy. NSI faculty members' research covers a range of central and autonomic nervous system functions, from sensation to neuronal plasticity, memory, motor control, and disease and injury processes. Their research uses a full range of experimental approaches, from molecular and cellular studies to neuroprosthetics, systems neuroscience, and mathematical modeling. The purposes of this training program are to help postdoctoral fellows develop strong research skills in multiple neuroscience disciplines, develop critical thinking and scientific communication skills, develop a scientific knowledge base in their area(s) of specialization, acquire technical and laboratory management skills needed to direct their own independent research programs, and develop a publication record - all of which are essential for ultimately establishing and maintaining an independent career in neuroscience research. In addition, NSI stresses community outreach as well as the application of one's basic research to health-care problems. As a result, the training program is designed to instill an awareness that the purpose of scientific research goes beyond the exploration of the unknown, to include education and application. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Congenital stationary night blindness (CSNB) is a group of non-progressive retinal diseases characterized by impaired scotopic vision. Mutations in a number of genes have been shown to be associated with CSNB. They generally affect synaptic transmission between photoreceptors and the second order bipolar cells. When the mutant gene is expressed only in rods, or rod bipolar cells, the phenotype is limited to night blindness. However, when the mutant gene function is also required for synaptic transmission between cones and cone bipolar cells, further visual symptoms are apparent such as myopia, hyperopia, nystagmus, and reduced visual acuity. One such example are mutations in GRM6, the gene encoding mGluR6, which cause an autosomal recessive form of CSNB. In the retina, visual information is 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 decreases in response to light. Two types of postsynaptic cells, the ON- and OFF-bipolar cells, respond with opposite polarity to glutamate released by photoreceptors, thus establishing the opposing visual pathways. The basis of signaling in OFF-bipolar cells, which relies on the activation of ionotropic glutamate receptors, is well understood. The signaling pathway that generates the light response in ON-bipolar cells, however, is more complex, and the molecular mechanisms remain to be elucidated. The ON-bipolar cell signaling pathway originates with a unique metabotropic glutamate receptor, mGluR6, which is found on the dendrites of ON-bipolar cells. mGluR6 acts via a G-protein, Go, to regulate the activity of an unidentified cation channel such that the light-induced decrease in synaptic glutamate opens the channel and depolarizes the cell. Recently, it has been reported that CSNB in Appaloosa horses is associated with a mutation causing a reduced expression of the TRPM1 cation channel. We hypothesize that TRPM1, and possibly other related TRP channels, are the cation channels coupled to mGluR6 that mediate the depolarizing light response of ON-bipolar cells. We further suggest that mutations in TRP channels will cause CSNB. Using a combination of biochemical, immunohistochemical, and electrophysiological approaches, we will test this hypothesis by answering the following questions: 1. Which TRP channel variants are expressed in ON-bipolar cells? 2. Do mice that carry null mutations in TRP channels expressed in bipolar cells have CSNB? 3. Can the physiological and pharmacological properties of retinal ON bipolar cell responses be reproduced in transfected HEK cells expressing the proper combination of TRP channel variants? 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: Congenital stationary night blindness (CSNB) is an inherited eye disorder causing night blindness, and also often shortsightedness, nystagmus (involuntary eye movement), and reduced visual acuity, even under normal lighting conditions. The proposed research will determine if abnormal ion channel function in the eye can be a cause of CSNB.

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.

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.