Judith Mosinger Ogilvie, PhD - US grants

Trophic interactions among sensory cells, neurons and supporting cells play an important role in the development and maintenance of sensory function. Loss of any one component may lead to atrophy of other cells, resulting in sensory deficit. Photoreceptor loss is the primary cause of visual impairment in several retinal degenerative diseases such as retinitis pigmentosa and macular degeneration. Sensory impairment resulting from degeneration of neural tissue was once considered irreversible. New possibilities are being raised as understanding of biochemical and molecular mechanisms of photoreceptor degeneration increases in animal models such as the rd (retinal degeneration) mouse. Furthermore, recent neural transplantation efforts related to degenerative brain diseases such as Parkinson's, Huntington's and Alzheimer's have stimulated research on neurosensory transplantation. Recently, this lab has presented evidence that normal photoreceptors transplanted from healthy mice can rescue cone photoreceptors in the rd mouse. This is the first instance of neurosensory rescue in an animal model relevant for a human sensory dystrophy. This proposal is based on the hypothesis that normal rod photoreceptors have a trophic effect on cone photoreceptors and supporting cells such as retinal pigment epithelium (RPE). The long term goal of this research is to develop an understanding of the molecular changes that underlie the secondary degeneration of cones and RPE in the rd mouse and to develop methods to prevent or minimize this loss. To achieve these goals, (I) normal mouse photoreceptors will be transplanted into the rd mouse followed by statistical and morphometric analysis to measure the effect of the transplanted cells on the survival of host cells, (2) an organ culture system will be developed with normal and dystrophic retinal tissue to simulate the in vivo model and (3) the organ culture system will be manipulated with trophic factors known to mediate sensory cell growth to better understand what molecular mechanisms are involved in maintenance and survival of sensory and supporting cells. The proposed re-entry training program builds on previous training by reestablishing both current knowledge of retinal research and technical expertise in light and electron microscopy. New training will emphasize knowledge of neurotrophic factors and cell and organ culture techniques. These elements will be use to build a research program focusing on trophic interactions between neural and support cells in the visual system.

DESCRIPTION (provided by applicant): The rod photoreceptors of the rd1 mouse degenerate in vivo or in organ culture by one month of age as a result of a defect in the beta-subunit of the cGMP-phosphodiesterase gene. As with most models of retinitis pigmentosa, the underlying mechanism of degeneration remains poorly understood. Dopamine is a neuromodulator affecting most, if not all, cell types in the vertebrate retina. We have discovered that dopamine antagonists from either the D1- or D2-receptor families completely block the degeneration of photoreceptors in the rd1 retinal organ culture model. Current theoretical models and observations of dopaminergic function fail to explain this surprising result. For example, only the D4 receptor subtype has been clearly identified in mouse photoreceptors; yet a D1-family antagonist is equally protective. Also, D1- and D2-family receptors generally act through opposing pathways to modulate the effects of the other; here they give the same result. Finally, dopamine generally has subtle, modulatory effects in the retina; here the effect on cell survival is dramatic and complete - no morphological difference can be detected between wild type and treated rdl organ cultures after 4 weeks, when nearly all of the rods have degenerated in the untreated rd1 culture. We propose experiments to address these differences and to test the significance of our findings in vivo. First, we will determine whether the absence of dopamine receptors can increase photoreceptor survival and function in the rd1 mouse retina in vivo. Secondly, we will address two aspects of the underlying mechanism, first testing a novel hypothesis concerning dopamine receptors, a subtype of the larger G-protein-coupled receptor family. In a recent paradigm shift, G-protein-coupled receptors, previously thought to function only as monomers, are now recognized to sometimes form heterodimers with atypical pharmacology and function. Such novel characteristics have recently been demonstrated with opioid, GABA, and other GPCRs. The formation of heterodimers comprised of different dopamine receptor subtypes could explain many of the perceived incongruities. We will also determine if D1-family dopamine receptors are present in rd1 photoreceptors. The proposed experiments will generate two important results. First, we will determine the importance of dopamine signaling in photoreceptor degeneration in vivo, which could lead to entirely new therapeutic approaches for retinal degeneration. Secondly, we will investigate the underlying mechanism including testing a dopamine receptor heterodimer-induced model of degeneration which could lead to a new area of investigation in retinal cell biology.

DESCRIPTION (provided by applicant): Photoreceptors are highly specialized ciliated epithelial cells that are easily accessible and undergo cell differentiation postnatally. The long-term goal of our research program is to understand the molecular signaling pathways underlying vertebrate photoreceptor development and cell differentiation, with particular focus on how disruption of these pathways leads to early onset vision loss. The objective of this proposal is to determine the role of the protein PRA1 in rod photoreceptor cell differentiation. During development, precursor photoreceptor cells exit the cell cycle and undergo a transformation to produce highly polarized cells with unique sensory specializations. Although morphological differentiation of photoreceptors is well characterized, less is known about the underlying molecular mechanisms. The rd1 mouse retina, characterized by early-onset, rapid degeneration of rod photoreceptors, was the first identified animal model of retinitis pigmentosa. Interestingly, rd1 rods show developmental defects, including failure to undergo normal synaptogenesis and outer segment differentiation, significantly before degenerative changes appear, presenting a model for investigations of photoreceptor development as well as a congenital neuronal disease. In preliminary studies, we have performed microarray analysis of wild type mouse retina compared to rd1 mutant retina at P2-8, prior to onset of photoreceptor apoptosis. Only two genes were consistently and significantly downregulated in the rd1 mouse at all time points: PDE6b, the mutant gene, and Prenylated rab acceptor 1 (PRA1). Small GTPases in the Rab family are known to be critical for vesicular trafficking involved in maintenance of both the outer segment and the synapse in the mature photoreceptor. PRA1 regulates delivery of Rab GTPases to membranes in eukaryotic cells, although its precise function and mechanism of action are poorly understood. We hypothesize that PRA1 regulation of vesicular trafficking is necessary and sufficient for synaptic and outer segment differentiation in rod photoreceptors. We propose to test our hypothesis with the following specific aims: (1) Determine the expression pattern of PRA1 in the adult and developing wild type and rd1 mouse retinas using Western blot and immunohistochemistry, and (2) determine whether PRA1 is necessary and sufficient for synaptic and outer segment differentiation in rod photoreceptors using RNA based loss-of-function and gain-of-function techniques. The proposed work is innovative in being the first to identify the significance of PRA1 in an animal model of neuronal development and disease. Upon completion of this work, we expect to have determined whether PRA1 expression can restore normal rod photoreceptor cell differentiation in the rd1 mouse retina. These results are ultimately expected to have a significant impact on the field of developmental neurobiology and to provide insights into mechanisms underlying aberrant differentiation, leading to improvement in childhood visual health. PUBLIC HEALTH RELEVANCE: The proposed experiments will elucidate mechanisms involved in development and differentiation of a highly specialized neuronal epithelial cell type: the rod photoreceptor cell. The rd1 mouse model of juvenile retinitis pigmentosa will be used to gain insights into the source of developmental aberrations. Greater understanding of these mechanisms could ultimately lead to targeted therapeutic interventions for juvenile retinitis pigmentosa and improved childhood visual health.