Susan E. Brockerhoff - US grants

Investigations of the cellular organization and composition of the retina have provided an understanding of how humans visualize their surroundings and a framework necessary to cure human retinal diseases. Nevertheless, many questions about vertebrate retinal function remain unanswered. For example, although many of the components involved in cone photoreceptor light responses have been identified, the precise mechanisms by which these molecules function to define cone physiology as distinct from rod physiology are unclear. Also, although different cone types in the vertebrate retina are thought likely to express unique molecules, few such molecules have been identified. Identifying cell- type specific molecules would answer questions both about the formation of specific connections within the retina and questions about the establishment of cell-type position and identity. The goal of this proposal is use zebrafish mutants to answer these questions. We have developed a behavioral assay that efficiently identifies zebrafish mutants with subtle and specific defects in retinal function. 1). We will continue to isolate more mutations as a resource for understanding vertebrate retinal function. 2) We will characterize mutations in detail using biochemical, molecular and physiological approaches. We will focus initially on three mutations. Two of these, noa and nrb, produce abnormalities in cone photoreceptor physiology, and the third mutations, pob, causes the selective loss of red cone photoreceptors within the retina. Our characterization will help define the physiological responses of cone photoreceptors. 3.) We will also use the pob mutation to identify molecules specific to red-sensitive cone photoreceptors. 4). Finally, we will define the molecular nature of the defect in noa, nrb and pob by using a candidate gene or positional cloning approach. As a first step towards this end, we will generate a database of retina-specific genes and map these genes.

DESCRIPTION (provided by applicant): Vertebrate cone photoreceptors are responsible for color and daytime vision. They have been extensively studied and much is known about their structure and function. For example, anatomical studies have described the morphologies of cone cells, the types of synpases they form and how they can adapt their shape in response to environmental and circadian factors. Furthermore, electrophysiological studies have described the physiological properties of cone responses to brief flashes of light and to steady illumination. Nevertheless there are many unanswered questions about cone structure and function. For example, although the physiological description of cone adaptation to illumination is well documented, the biochemical mechanism underlying this response is unknown. In addition, the mechanisms by which ribbon synapses form and are regulated are unknown. Finally many factors that influence cone photoreceptor shape and viability have yet to be discovered. In this proposal the investigators use zebrafish to identify genes essential for normal cone structure and function. The proposal relies heavily on the previous success of the investigator to identify genes essential for vertebrate cone function using a similar screening strategy. The specific aims of the proposal are: Specific Aim#l: Identify recessive mutations that are essential for normal cone photoreceptor function using an OKR behavioral screen followed by ERG analysis. Specific Aim#2: Perform histological analyses of the retinas of mutants with defects in cone photoreceptor function. Specific Aim #3: Identify the mutated genes by positional cloning and candidate gene analysis. All resources generated from this proposal will be made available to the scientific community. A specific plan for distributing the resources generated from this proposal has been designed with investigators at the zebrafish resource center in Oregon. This plan is presented as part of the proposal.

DESCRIPTION (provided by applicant): We have identified a blind zebrafish mutant with rapid degeneration of cone photoreceptors due to a mutation in the cone phosphodiesterase c (pde6c) gene, a key regulatory component in cone phototransduction. We will use this mutant combined with the advantages of the zebrafish model system to answer two fundamental questions in photoreceptor biology. 1. What are the cell-type and cell density requirements that lead to the death of healthy photoreceptors in the presence of dying photoreceptors (the bystander effect)? 2. What is the molecular pathway leading to cone degeneration in the absence of phosphodiesterase? Zebrafish is a vertebrate model system that provides several advantages for studying retinal degeneration. In this case, the loss of PDE6c in zebrafish provides a unique opportunity to gather new information about retinal degeneration. Our current understanding of the causes of photoreceptor degeneration is not sufficient to develop successful therapies. Zebrafish develop rapidly and have a well-characterized retina. Furthermore, they are optically clear, can be used efficiently in genetic screens and, importantly, can be made mosaic easily through cell transplantation. These features will enable us to go beyond studies begun in other systems. New information from the zebrafish model will enhance our understanding of retinal degeneration and aid in the development of therapies. We plan to take advantage of our detailed understanding of the photoreceptor's phototransduction cascade and the genetic and molecular tools unique to zebrafish to determine the molecular trigger(s) stimulating (Aim #2) and preventing (Aim #3) degeneration. We also will use our cone degeneration mutant to make mosaic retinas containing mixtures of mutant and non-mutant cones to dissect parameters essential for causing the bystander effect (Aim #1). PUBLIC HEALTH RELEVANCE: Zebrafish is a vertebrate model system that provides several advantages for studying retinal degeneration. We have identified a blind zebrafish mutant with rapid degeneration of cone photoreceptors due to a mutation in the cone phosphodiesterase c gene (pde6c), a key regulatory component in cone phototransduction. New information from the study of the zebrafish mutant pde6cw59 will enhance our understanding of retinal degeneration and aid in the development of therapies to treat this common inherited form of blindness.

Photoreceptors are highly polarized, compartmentalized cells. Protein synthesis initiates in the inner segment and then transport proceeds in the apical direction toward the outer segment or basally toward the synapse. In recent years, several investigators have made seminal discoveries about the mechanism(s) of transport to the outer segment. In contrast, very little is known about the transport of proteins destined for the synapse or the sorting within the inner segment of proteins destined for different cellular compartments. Phosphoinositides are known to be key regulators in membrane trafficking and are involved in signaling, specification and protein recruitment in a wide variety of cell types. A key regulator of cellular phosphoinositides is the lipid phosphatase, synaptojanin I (SynJ1), whose primary intracellular target is PI(4,5)P2. We have established zebrafish as a model system in which to evaluate both phosphoinositide signaling and SynJ1 function in the process of protein sorting in cone photoreceptors. Our current work finds a role for SynJ1 in the inner segment. We find that SynJ1 concentrates in the cone inner segment and that large vesicles abnormally accrue and/or the Golgi architecture is disrupted in nrc mutants lacking this protein. We hypothesize that the inner segment phenotype reflects a disruption of transport and sorting of proteins destined for the synapse. We propose a series of experiments that test this hypothesis and define precisely the abnormal vesicular structures we detect in nrc inner segments, their content and derivation. The specific expected outcome of our proposal is a detailed understanding of the inner segment defect detected in nrc when the balance of polyphosphoinositides within the photoreceptor is disrupted due to the loss of the critical PI(4,5)P2 phosphatase, SynJ1. In addition, our studies will provide fundamental information about the cellular distribution of polyphosphoinositides in both wild-type and nrc cone photoreceptors. Finally, our studies will define the importance of different structural domains of SynJ1. The critical, fundamental information we discover from the studies outlined in this proposal will help open this field to many additional important investigations.

? DESCRIPTION (provided by applicant): Intracellular crosstalk mediated by calcium ions (Ca2+) is fundamentally important for understanding regulation of cellular function. Cellular Ca2+ fluxes and mitochondrial Ca2+ dynamics, influence the activity of mitochondrial enzymes involved in energy metabolism and cellular redox. These changes influence post-translational modification of proteins in mitochondria, the cytoplasm and the nucleus, which control cell functions such as energy metabolism and transcription. We hypothesize that Ca2+ signaling by mitochondria in photoreceptors is critical for their function and survival. The study proposed here will define how photoreceptor activity is regulated by mitochondrial Ca2+ to meet the specialized demands of this unique sensory neuron. Using genetically encoded Ca2+ indicators and in vivo imaging, we have documented large Ca2+ fluctuations in the cell bodies and mitochondria of zebrafish cone photoreceptors. We also developed methods for manipulating Ca2+ in cell bodies and mitochondria and for detecting protein modifications and cellular redox changes. We will use these methods to determine how Ca2+ fluxes within photoreceptors are processed by mitochondria. We will study how mitochondria regulate Ca2+ in photoreceptor cell bodies and the role of the mitochondrial Ca2+ uniporter (Aim 1). We also will determine how Ca2+ controls the cytoplasmic redox potential and acylation of proteins (Aim 2).

ABSTRACT Aerobic glycolysis dominates energy metabolism in photoreceptor neurons. In aerobic glycolysis, most of the glucose a cell consumes gets converted into lactate, even when O2 is present. Aerobic glycolysis has been linked to anabolic activities that support robust growth in cancer cells. This has led to the hypothesis that enhancing aerobic glycolysis will make photoreceptors more robust and resistant to stresses caused by genetic deficiencies. Recent work supports this hypothesis. The transcription repressor Sirtuin 6 (Sirt6) has several activities, one of which is to repress expression of glycolytic genes. Sirt6 deficiency increases expression of key metabolic enzymes that carry out aerobic glycolysis. Remarkably this slows degeneration of rods and cones caused by a PDE6b mutation. The project in this proposal will identify metabolic gene{s) that most efficiently enhance photoreceptor survival. We will use zebrafish as a model organism. Specifically, we will increase expression in cones of key metabolic enzymes that enhance glycolytic activity and assess, using imaging strategies we developed, the slowing of degeneration caused by PDE6-deficiency. Initially, we will focus on enzymes most likely to be rate-limiting; Glut-1, hexokinase, pyruvate kinase, phosphofructokinase, pyruvate dehydrogenase kinase and lactate dehydrogenase (Aim 1). We then will use metabolomics to identify specific metabolic changes that are linked to prolonged photoreceptor survival (Aim 2). Overall, our studies will provide basic metabolic information needed to understand how enhancing aerobic glycolysis can promote photoreceptor survival. Future studies based on these findings will be aimed at developing therapeutic strategies that enhance metabolic features that promote photoreceptor viability.

Project Summary Many different genes and mutations have been associated with photoreceptor degeneration. This enormous genetic heterogeneity requires the development of treatment approaches targeting common mechanisms and pathways, which can effectively treat a condition regardless of the underlying genetic cause. Mutations in several genes involved in guanine nucleotide homeostasis lead to photoreceptor degeneration, likely due to the unique metabolic demands for cyclic GMP in photoreceptor signaling. Despite this critical importance, basic aspects of regulation of purine metabolism in photoreceptors have not been investigated rigorously. This proposal is focused on the critical role of IMP dehydrogenase 1 (IMDPH1), the enzyme that regulates flux through the parallel de novo adenine and guanine nucleotide biosynthesis pathways. Nine different missense mutations in IMPDH1 are associated with dominant forms of retinitis pigmentosa or Lebers congenital amaurosis, each with varying severity of disease phenotype. None of the mutations has a direct effect on the intrinsic biochemical activity of the enzyme, and the mutations do not have deleterious effects in tissues other than the retina. For many years progress has been stalled on the molecular mechanism of IMPDH1-induced retinopathies due to a lack of animal models to study the disease pathology, and the lack of a clear defect at the enzyme level. IMDPH forms micron- scale dynamic filaments both in vitro and in vivo in response to increased demand for guanine nucleotides. We have now discovered that the IMPDH1 retinal degeneration mutations have a direct effect on the filament form, either promoting constitutive assembly or preventing polymerization entirely, with concomitant defects in allosteric regulation of the enzyme activity. Here, we propose a collaborative and initial effort to define the structure, function, and metabolic role of IMPDH1 in healthy photoreceptors and in transgenic animals that express IMPDH1 mutant alleles associated with photoreceptor degeneration. Our multidisciplinary approach combines cryoEM structural analysis with imaging and metabolic measurements in intact retinas. Our work will provide novel insight into the unique role of IMPDH1 in photoreceptors, and lay the groundwork for analyzing more mutations and for future targeted therapies to prevent blindness due to cell death.