Jeannie Chen - US grants

The objective of this proposal is to use transgenic mice to understand the normal function of recoverin and to test whether its dysfunction will lead to retinopathy. A large body of evidence points to calcium as an important messenger in reestablishing the dark steady state, and recent biochemical analyses demonstrate that recoverin is a mediator of this calcium response. The physiological function of recoverin will be probed by introducing gain or loss of function into transgenic mice, and by introducing biochemically defined mutants into transgenic mice with disrupted recoverin genes. Specifically, targeted recoverin overexpression (gain of function) in the photoreceptor layer of transgenic mice will be achieved by using a characterized rhodopsin promoter to drive expression of the recoverin cDNA. Targeted gene disruption (loss of function) will be achieved by making homologous recombination in pluripotent embryonic stem (ES) cells and to use these cells to generate transgenic mice bearing disrupted recoverin genes. Recoverin mutants with defined biochemical defects will be introduced into transgenic mice with a null background, i.e. mice generated from the ES cells, to study specific mutant effects on phototransduction. The studies proposed here will contribute toward a better understanding of normal recoverin function and may also shed light on other visual processes which may be under calcium control. Interestingly, recoverin is the target antigen for autoantibodies formed in patients with caner-associated retinopathy (CAR). Significantly, sera from five out of five CAR patients tested contained antibody against recoverin. This striking correlation emphasizes the need to study the physiological role of recoverin in normal visual function and in retinopathies.

The objective of this proposal is to use transgenic mice to understand the normal function of recoverin and to test whether its dysfunction will lead to retinopathy. A large body of evidence points to calcium as an important messenger in reestablishing the dark steady state, and recent biochemical analyses demonstrate that recoverin is a mediator of this calcium response. The physiological function of recoverin will be probed by introducing gain or loss of function into transgenic mice, and by introducing biochemically defined mutants into transgenic mice with disrupted recoverin genes. Specifically, targeted recoverin overexpression (gain of function) in the photoreceptor layer of transgenic mice will be achieved by using a characterized rhodopsin promoter to drive expression of the recoverin cDNA. Targeted gene disruption (loss of function) will be achieved by making homologous recombination in pluripotent embryonic stem (ES) cells and to use these cells to generate transgenic mice bearing disrupted recoverin genes. Recoverin mutants with defined biochemical defects will be introduced into transgenic mice with a null background, i.e. mice generated from the ES cells, to study specific mutant effects on phototransduction. The studies proposed here will contribute toward a better understanding of normal recoverin function and may also shed light on other visual processes which may be under calcium control. Interestingly, recoverin is the target antigen for autoantibodies formed in patients with caner-associated retinopathy (CAR). Significantly, sera from five out of five CAR patients tested contained antibody against recoverin. This striking correlation emphasizes the need to study the physiological role of recoverin in normal visual function and in retinopathies.

DESCRIPTION (Adapted from applicant's abstract): The highly invariant single photon response is a salient feature of the photoreceptor. The single photon response is shaped by the kinetics of the amplification cascades as well as the kinetics of deactivation. Although the steps in the amplification pathway are well-elucidated, mechanisms that regulate signal deactivation are still poorly understood. In vitro reconstitution experiments have identified rhodopsin phosphorylation and arrestin binding to be important in rhodopsin inactivation. However, the physiological relevance of these processes in signal inactivation is uncertain because of difficulties in duplicating physiological conditions and to follow real time reaction kinetics in reconstitution experiments. To overcome these limitations, transgenic mouse technology is used to target mutations in the component of the photocascade. Photoreceptors from these transgenic mice then become a resource for single cell recording and biochemical assays. They will use such an approach to investigate mechanisms regulating rhodopsin deactivation. Specifically, they will examine how phosphorylation leads to receptor inactivation in vivo by measuring the kinetics of signal termination in mice expressing a variety of rhodopsin mutants that lack phosphorylation sites at the COOH-terminus. They will dissect and compare the functional roles of arrestin and its COOH-terminal truncated variant by expressing arrestin alone or the truncated isoform alone in the background of arrestin knock-out mice. They will test the hypothesis that in vivo, arrestin binding limits the extent of rhodopsin phosphorylation by rhodopsin kinase and dephosphorylation by the endogenous protein phosphatase 2A. Finally, they will investigate the process of long-term light adaptation (cellular and molecular changes that develop over days to weeks) in the arrestin knock-out mice where constitutive phototransduction is occurring, and to see whether unabated signal flow is a cause for some forms of retinal disorders. The transgenics approach serves to interface biochemistry, electrophysiology and morphology and brings together a multi-disciplinary effort to investigate mechanisms regulating phototransduction. Their findings will also have relevance to other G-protein signaling pathways.

DESCRIPTION (provided by applicant): The broad goal of this proposal is to decipher the molecular mechanisms of light adaptation in both rod and cone photoreceptor cells. It has been known for some time that calcium orchestrates several feedback mechanisms that serve to prevent signal saturation in retinal photoreceptors. Thus far, three Ca 2+-dependent steps in the phototransduction cascade of rods have been identified in vitro; namely, rhodopsin kinase (RK) activity, guanylate cyclase (GC) activity, and affinity of cGMP-gated (CNG) channel for cGMP. The Ca2+ dependence of these activities is conferred by Ca2+-binding proteins: recoverin, guanylate cyclase activating proteins (GCAPs), and calcium calmodulin (Ca2+-CaM), respectively. The manner by which Ca2+ regulation of these enzymatic steps ultimately translates to cellular adaptation behavior is not fully understood. Since the overall ability of the cells to adapt to light very likely reflects the summation of individual Ca2+-sensitive transduction steps, a complete understanding of the molecular basis of adaptation will rely upon an experimental design that allows for specific isolation and quantitative assessment of their individual contributions in intact photoreceptors. Toward the attainment of this goal, we have specifically disrupted Ca2+ feedback to RK and GC by targeted disruption of recoverin and GCAPs, respectively, in transgenic mice; their contribution to the ability of rods to adapt to light has been evaluated by suction electrode recordings. We now propose to continue the study of rod adaptation by assessing the contribution of Ca2+ feedback to the CNG channel, and to use quantitative electroretinographic analysis in order to investigate the means by which the same Ca2+ feedback regulations control light adaptation in cone photoreceptors. Our established Specific Aims are to: 1) Test the hypothesis that Ca2+ modulation of the CNG channel's affinity for cGMP contributes to the ability of rods to adapt to light. 2) Test the hypothesis that recoverin has little effect on the first phosphorylation events but delays phosphorylation during the recovery phase of the light response. 3) Test the hypothesis that GCAPs regulate sensitivity adjustment in cone photoreceptors. 4) Test the hypothesis that recoverin regulates cone PDE adaptation. These studies will help us understand the molecular mechanisms behind some of the fundamental differences between rod and cone photoreceptor cells during light adaptation.

[unreadable] DESCRIPTION (provided by applicant): Retinal rods and cones utilize visual pigments that are coupled to G-protein signaling cascades to detect the presence of photons. Little is known about how these two cell types use such a remarkably similar phototransduction cascade to achieve their distinct functional properties. A major objective of this project is to address this fundamental question. An understanding of the mechanisms that regulate phototransduction also has important disease relevance because deregulated signaling that occurs at different transduction steps often has a negative impact on photoreceptor cell survival. Our second objective is to apply what we learned in phototransduction and translate that knowledge into a better understanding of disease mechanisms in rods and cones so that a rational therapeutic strategy can be devised. In the first aim, we will test the hypothesis that differences in the functional properties between rods and cones can be explained, in part, by the transduction efficiency between the visual pigment and transducin, the visual G-protein. This hypothesis is supported by our preliminary results that show a 100-fold decrease in sensitivity when cone transducin was placed downstream of rhodopsin. Experiments in Aim 1 will systematically analyze the contribution of the rod and cone isoforms of the heterotrimeric transducin subunits toward the observed decrease in transduction efficiency at this step. The potential function of the G[unreadable]? subunit in controlling photoreceptor noise and response recovery will also be investigated. In the second aim, we will investigate the cell death pathways that are triggered by light exposure or genetic mutations that lead to "equivalent light". Experiments in Aim 2 are designed (a) to probe the underlying mechanism for the toxicity of the rhodopsin/arrestin complex; (b) to investigate whether endocytosis of the rhodopsin/arrestin complex is required to generate the cell death signal; (c) to analyze whether constitutive phototransduction in cones is a potential mechanism for cell death; and (d) to study the involvement of ATF-3 and ATF-4 transcriptional regulators in the cellular response to light damage. The outcome from these experiments will address the fundamental question as to how cells may utilize G-protein signaling cascades, which consist of highly similar protein members, to achieve diverse signaling properties. In addition, we will gain a better understanding of the relationship between defective signaling and disease. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The long-term objective of our research is to elucidate the molecular mechanisms underlying the function of nicotinic acetylcholine receptors (AChR). The muscle AChR is a ligand-gated ion channel that mediates fast signal transmission at the neuromuscular junction (NMJ). The receptor protein was first purified nearly two decades ago, but its atomic structure remains poorly defined. With the support of our previous NIH grant, we have identified the minimal ligand-binding domains on the alpha and delta subunits of mouse muscle AChR. In addition, we have established a yeast expression system that allows the production of large quantities of the extracellular domains of x subunit in monomeric form (alpha211) as well as the extracellular domains of both alpha and delta subunits in dimeric form (alphadelta heterodimer). Biochemical and pharmacological studies have demonstrated that the recombinant proteins fold in native-like conformation with high affinity to cholinergic ligands. Furthermore, we have optimized the conditions for uniform isotopic labeling and structural determination of o211 by nuclear magnetic resonance (NMR). Initial screening of conditions for crystallization has led to the production of small crystals of alphadelta heterodimers in complex with acetylcholine (ACh). As the logical extension of our past studies, the specific aims of research proposed in this competing renewal application are: (1) to determine the high-resolution structure of o211 by multidimensional NMR; and (2) to solve the atomic structure of o2i heterodimer in complex with ACh or the autoimmune antibody mAb35 by Xray diffraction. Elucidation of the 3D structure of AChR holds the key to understanding how the receptor interacts with ligands. Such information may also be applicable to studies of other ligand-gated ion channels including GABA, glycine and 5-HT3 receptors. As these proteins play a role in the pathogenesis of pain, dementia, epilepsy, and stroke determination of their structure is essential for the rational design of more selective therapeutic agents. The third specific aim of our research is to study the structure of neural agrin, a protein secreted by motoneurons that induces clustering of AChRs on muscle cell membrane at the NMJ. Multiple forms of agrin that differ in binding properties and bioactivity are generated through alternative splicing of agrin mRNAs in a variety of tissues. How alternative splicing regulates AChR-clustering activity is completely unknown. Here, we propose to solve the solution structure of the C-terminal domains of both neural and muscle agrins by NMR. The structural information will help to recover the molecular mechanisms underlying the important function of agfin during synaptogenesis at the NMJ.

The control of muscle contraction and human movement relies on the establishment of precise connections between motor neurons in the brain and spinal cord and the skeletal muscles. During embryonic development, motor nerves make contact with muscle cells and induce the muscle membrane to form a specialized domain called the neuromuscular junction. At the neuromuscular junction, many protein molecules including the nicotinic acetylcholine receptor, rapsyn, utrophin, and dystroglycans, are highly concentrated. These molecules are assembled into a large complex, termed the postsynaptic apparatus. The postsynaptic apparatus is critically important for the transmission of brain signals from motor nerves to muscles, and for the maintenance of normal muscle structure and function. Disruption of the protein complex is known to cause many neuromuscular diseases including myasthenia gravis, muscular dystrophy, and etc. The principal investigator of this research project will investigate the molecular mechanism that governs the development of postsynaptic apparatus at the neuromuscular junction. He will test the hypothesis that both the formation and maintenance of the postsynaptic protein machinery are critically dependent upon a recently identified molecule, called Adenomatous Polyposis Coli (APC). APC acts as a scaffold to organize and stabilize the membrane protein complex by tethering it to the cytoskeleton at neuromuscular junction. Two sets of experiments have been proposed. First, a genetic approach to determine whether deletion of the APC gene is detrimental to the structure and function of neuromuscular synapse in mouse embryos will be performed. Second, the question whether the APC protein has a physiological role in the development and maintenance of nerve-muscle connections in postnatal and adult life will be examined.

The research will create unique opportunities for postdoctoral fellows, graduate and undergraduate students to learn and study the process of how neurons in the brain make precise connections with their target tissues during development. The novel concepts, approaches, and techniques of the projects will be taught in various summer programs to train high school and minority students for advanced biological research.

DESCRIPTION (provided by applicant): Myasthenia gravis is a neurological disorder characterized by weakness and fatigability of the skeletal muscles. Autoimmune antibodies against the nicotinic acetylcholine receptor (AChR) at the neuromuscular junction (NMJ) are known to be the main cause of the disease. The AChR is a ligand-gated ion channel that mediates the transmission of electric signals from motor nerve to muscle. About two-thirds of autoimmune antibodies from sera of patients with myasthenia gravis bind to the Main Immunogenic Region (MIR), an epitope located on the extracellular domain of the alpha subunit of AChR. The antibodies cross-link the AChRs on muscle cell membrane, resulting in an increased rate of internalization/degradation of the receptor and impaired neuromuscular transmission. Current treatments for myasthenia gravis can only temporarily relieve symptoms. They are usually non-specific and cannot change the cause of the disease. The long-term goal of our research is to identify new drugs that can act more effectively with fewer side-effects for myasthenia gravis. In this R21 grant application, large quantities of the N-terminal, extracellular domain of alpha subunit of human muscle AChR will be generated in a yeast expression system. The receptor fragment will be crystallized in complex with mAb35, a reference monoclonal antibody that can cause experimental myasthenia gravis. The three-dimensional structure of the protein complex will be determined by x-ray diffraction. The structural information will be key to rational design of drugs that can prevent binding of autoimmune antibodies to AChR. PUBLIC HEALTH RELEVANCE This study should provide new insight into the molecular structure of a complex formed between the nicotinic acetylcholine receptor and autoimmune antibodies that causes myasthenia gravis, a relatively common neurological disease. The coordinates of the protein crystal structure will aid greatly the rational design of more potent and specific drugs as well as for understanding the role of a particular biochemical process in the development of myasthenia gravis. If successful, the exploratory study outlined in this R21 grant application may open a new avenue for future drug discovery aimed at effective control of the disease in patients.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Phototransduction is a signaling cascade whereby photon absorption is converted into a change in electrical current. Members of the phototransduction cascade are abundantly present in the thin outer segment compartment of the rod photoreceptor. Many of these proteins have been crystallized and high resolution structures have been deduced. However, it is not known how these proteins are assembled. The objective of this proposal is to obtain a detailed understanding of the signaling complexes in the vertebrate rod photoreceptor. The Chen laboratory has made a number of transgenic mice with targeted deletion of these proteins. Tomography of outer segments from these mice may be helpful in the reconstruction of the protein complexes involved in phototransduction.

? DESCRIPTION (provided by applicant): Retinal rods utilize a prototypical G-protein signaling cascade to encode our visual scene under dim light. Over-stimulation of this cascade by bright light, or genetic mutations that act as equivalent light, are known environmental factors that exacerbate disease progression for age-related macular degeneration (AMD) as well as other retinal disorders in humans. Although it is known that rhodopsin activation is required for light-induced pathogenesis, the distinct molecular pathways remain to be defined. The first two aims will investigate biochemical reactions in rods that may slow dark adaptation following bright light exposure. The third aim will investigate two different mechanisms of light-induced rod cell death. The long-term objective of this proposal is to understand phototransduction in normal function and dysfunction so that this knowledge can be used to devise strategies for the treatment of human visual disorders.

Project Summary Rod photoreceptor death is a significant cause of human blindness, and much research effort has been expended towards their rescue or replacement using gene or stem cell therapy. However, rod death is followed by secondary changes in the inner retina such as dendritic remodeling, cell migration, and rewiring. The extent to which this reorganization obstructs the potential for recovery of vision following photoreceptor rescue is not known. The objective of this proposal is to examine signal processing in the affected retina following the genetic rescue of rods. To this end, we have created a mouse model of retinal degeneration caused by loss of expression of the ?-subunit of the cyclic nucleotide-gated (CNG?1) channel, a model for autosomal recessive retinitis pigmentosa in humans. The novelty of this mouse model is that CNG?1 can be expressed from the endogenous locus in all affected CNGB1-/- rods upon tamoxifen(TX)-inducible Cre- mediated recombination, leading to rod rescue. This proposal utilizes this mouse line to determine the impact of rescuing rod function on retinal signal processing. In Aim 1 we will examine how the structure and function of rod photoreceptors recovers following the restoration of CNG?1 expression. In particular we will examine the extent of functional recovery when TM is administered in mice with increasing severity of retinal degeneration, as this may identify a critical window for the efficacy of rod recovery and halting further degeneration. In Aim 2 we will examine how the synapse between rods and their primary postsynaptic partner, rod bipolar cells, is reformed following rod rescue. Synapses between rods and rod bipolar cells form retinal circuits that regulate our night vision. Finally, in Aim 3 we will evaluate how rod rescue impacts the function of retinal ganglion cells, which are the sole conduit for signals from the retina to reach higher brain areas. The central hypothesis is that while light sensitivity will recover substantially with rod rescue, some deficits in retinal signaling will persist and worsen at late rescue ages due to secondary changes in retinal circuits. These studies will define the window of opportunity for therapeutic intervention and provide a foundation for future studies aimed at reversing the negative effects of neural remodeling.

ABSTRACT Calcium (Ca2+) is a ubiquitous signaling molecule that controls the function and survival of neurons. The disrupted Ca2+ homeostasis in a wide range of photoreceptor mutations is believed to cause cell death, retinal degeneration and blindness. In vertebrate photoreceptors, Ca2+ changes also modulate the shutoff of the phototransduction cascade to accelerate light response recovery and background adaptation. It is thought that the concentration of Ca2+ in the outer segments of vertebrate photoreceptors is controlled by a dynamic balance between influx via the cGMP-gated (CNG) channels and extrusion via cell-specific Na+/Ca2+, K+ exchangers (NCKX), NCKX1 in rods and NCKX2 in cones. However, the extent to which these exchangers control the Ca2+ homeostasis in mammalian photoreceptors and modulate phototransduction and cell survival has not been determined. In addition, it is not known whether other active or passive mechanisms for extruding Ca2+ are at play in the outer segments of mammalian rods and cones. We will perform experiments to establish the role of CNG and NCKX1 in regulating the Ca2+ homeostasis in mammalian rods and their effect on long-term rod survival and degeneration. We will also test the hypothesis that abnormal photoreceptor Ca2+ homeostasis mediates photoreceptor degeneration in a variety of blinding diseases and will determine the therapeutic potential of restoring the Ca2+ flux balance in photoreceptor channelopathies. We have identified NCKX4 as a second Na+/Ca2+, K+ exchanger expressed in mammalian cones. We will perform experiments to analyze the expression profile, morphology, and functional properties of NCKX2- and NCKX4- deficient mouse cones. These experiments will establish the molecular mechanisms for the efficient extrusion of Ca2+ from mammalian cone photoreceptors critical for the fast response kinetics and background adaptation of cones as our daytime photoreceptors as well as their effect on cone long-term survival and degeneration. Collectively, our experiments will establish the molecular mechanisms that mediate the extrusion of Ca2+ from mammalian photoreceptors. They will also help us understand the link between abnormal Ca2+ homeostasis and photoreceptor degeneration and might potentially lead to the development of treatments for channelopaties.

Project Summary ? Animal Models and In Vivo Imaging Core The Animal Models and In Vivo Imaging Core will meet the needs of participating investigators for using animal models of ocular diseases to conduct basic and translational vision research. The Core will provide access to advanced technologies and highly qualified technical support for generating and performing in vivo ocular imaging of animal models. The availability of animal models coupled with comprehensive in vivo tissue structure and function assessment will greatly accelerate study of molecular and cellular mechanisms underlying ocular disease processes, and facilitate translation of basic science findings for diagnostic and therapeutic applications. The specific aims are to provide instrumentation and technical expertise for: 1) generating and maintaining animal models of ocular diseases, performing surgical procedures, obtaining samples for genotyping, and harvesting ocular tissues; 2) enabling investigators to use various in vivo ocular imaging and tissue function testing technologies; 3) training investigators and research staff on the utility and operation of ocular imaging and function testing equipment; and 4) consultation regarding the appropriate animal models, surgical and imaging procedures to address research needs of participating investigators. .

PROJECT SUMMARY While amyloid plaques and neurofibrillary tangles are Alzheimer's disease (AD) defining features, Alzheimer himself originally identified a third pathology? inflammation of the brain's glial support cells. Neuroinflammation in AD is characterized by reactive astrocytes and microglia that surround amyloid plaques and chronically secrete inflammatory innate immune cytokines. The dominant view for decades has been that all forms of inflammation damage the AD brain. Yet, non-steroidal anti-inflammatory drugs failed to produce a positive signal for AD primary prevention. This raises a fundamental question: should we be blocking or possibly even promoting inflammation as an AD therapeutic? While the focus has mainly been on pro-inflammatory cerebral innate immunity, little attention has been paid to factors that curtail peripheral innate immune responses. The unifying theme of our work is that `rebalancing' peripheral innate immunity to homeostasis by releasing immunosuppression will limit AD progression. Strikingly, our focus on innate immunity in AD has just recently been validated by genome-wide association studies. These results have taken the field by storm; identifying clusters of AD risk alleles in core peripheral macrophage pathways. As a key cytokine suppressor of innate immunity and inflammation, transforming growth factor-beta (TGF-?) mRNA abundance is increased in AD patient brains. We hypothesize that the AD brain over- compensates to pro-inflammatory signals by producing these abnormally high levels of TGF-?. Paradoxically, this sets up early, low-level and chronic neuroinflammation that fails to support amyloid-? (A?) clearance. I and my team have shown in published and preliminary data that genetic or pharmacologic blockade of TGF-?- Smad 2/3 signaling in peripheral macrophages leads to brain entry of these cells and A? phagocytosis; sparing neurons from injury and restoring learning and memory. To further explore this theme, we have now generated the TgF344-AD rat that recapitulates cognitive impairment and the full array of human AD pathological features: neuroinflammation, plaques, tangles, and frank neuronal loss. In AIM 1, we will use non-invasive longitudinal imaging approaches to determine whether early neuroinflammation preempts later cognitive impairment, A? deposition, structural connectivity changes and neuronal death in TgF344-AD rats. AIM 2 is designed to longitudinally evaluate if blocking peripheral innate immune TGF-? signaling licenses A? phagocytosis and mitigates AD-like changes by delivering cutting- edge nanoparticles containing small molecule TGF-?-Smad 2/3 signaling inhibitor payload to hematogenous macrophages. Finally, we will pharmacologically delete peripheral macrophages to definitively establish if they are responsible for the beneficial effects of TGF-? signaling inhibition. While AD animal model studies are typically limited by cross-sectional designs, this project will break this barrier by coupling the most advanced multimodal, longitudinal brain imaging with peripheral TGF-? signaling inhibition in the TgF344-AD rat.