David R. Pepperberg - US grants

DESCRIPTION (provided by applicant): Exposure of rod photoreceptors to bright (rhodopsin bleaching) light triggers operation of the retinoid visual cycle and markedly desensitizes the rod flash response. Subsequent dark adaptation of the rods requires, in addition to excitation decay (shut-off of activated transduction intermediates and cGMP replenishment), recovery from "silent" desensitization processes that depress transduction signaling even after the near-complete recovery of circulating current. In vitro studies show that the magnitude of silent desensitization far exceeds that attributable to bleach-induced reduction in quantum catch, and have identified numerous likely contributing reactions. However, the lack of information on the operation of silent desensitization in the living eye is a major current obstacle to ultimately understanding in vivo mechanisms and relative roles of the contributing reactions. A main focus of the project is to determine the in vivo bleaching-dependence of the silent desensitizations's magnitude and timing in mouse and in human rods using paired-flash electroretinographic (ERG) recording, a recently developed technique that permits noninvasive determination of the full time course of the rod weak-flash response. In normally functioning rods of mice and of human subjects, and in abcr-/- mice that exhibit sluggish excitation decay due to lack of the ABCA4 transporter of all-trans retinal bleaching product, we will test the specific hypothesis that excitation decay rate-limits the duration of silent desensitization. The accuracy of the paired-flash method in quantitatively determining the rod flash response -- specifically, the possibility that ERG b-wave intrusion skews derivation of the rod response amplitude -- will be tested in experiments on the nob mouse, a b-wave deficient model. A further focus of the project is to test a recently raised hypothesis concerning retinoid metabolism in the retinal pigment epithelium (RPE), a process directly linked with the RPE's role in supporting rhodopsin regeneration in dark adapting rods. The hypothesis -- that 11-cis retinoid in the RPE regulates the efflux of all-trans retinol at the RPE basolateral membrane -- will be tested in radiolabeling experiments on mice that exhibit normal or impaired visual cycle operation. Results of the project will advance fundamental knowledge of rod dark adaptation and provide foundation for further studies of photoreceptor disease in human subjects and experimental animals.

DESCRIPTION: (Applicant's Abstract) Retinal degenerative diseases such as macular degeneration involve progressive dysfunction and deterioration of rod and cone photoreceptors. In these and other neurodegenerative diseases, neurons post-synaptic to the deteriorating cells are believed frequently to preserve their capacity for neural signaling; functional loss follows from the inability of the deteriorating pre-synaptic cell to stimulate the post-synaptic membrane receptor protein of a specific chemical synapse. Here we propose a novel project, the long-term goal of which is to restore stimulus-regulated signaling at nonfunctioning chemical synapses. The proposed approach is to develop nanoscale neuromodulating platforms (NNPs) that are responsive to external stimulating signals and interact physiologically with specific post-synaptic membrane receptor proteins. The essential feature of the platform is electrochemical control of the accessibility, to the receptor protein, of neurotransmitter derivatized and tethered to a signal-responsive substrate. In the first three years of the project we propose to construct and test prototype platforms (surface dimensions of ~0.1-1 mm) that, in response to light, modulate the electrophysiological activity of defined membrane receptor proteins (e.g., GABAc receptors) expressed in Xenopus oocytes. The platform's active surface will consist of alkyl and poly(ethylene oxide) chains that are covalently linked to the surface, derivatized with a ferrocene/ferricinium moiety, and distally terminated by an N-substituted analog of the relevant amino acid neurotransmitter. The analog's accessibility to the receptor protein will be regulated by an avalanche photodetector (APD) architecture of the platform, resulting in redox-control of the hydrophobicity of the ferrocene/ferricinium and a conformational change in the tethering chain.

DESCRIPTION (provided by applicant): Retinal degenerative diseases such as age-related macular degeneration are associated with dysfunction and deterioration of the rod and cone photoreceptors of the retina. Postsynaptic membrane receptor proteins of retinal neurons proximal to the rods and cones mediate the transmission of visual signals at multiple types of chemical synapses in the normally functioning retina, and there is reason to believe that these proximal retinal neurons in certain cases remain functional despite the disease-induced loss of rod and cone visual signaling. The long-term goal of the proposed project is to design and construct nanoscale molecular structures that can selectively attach to the extracellular face of specific membrane receptors of post-photoreceptor retinal neurons and, by modulating the postsynaptic receptor's activity in response to light, restore visual signaling in retina damaged by photoreceptor degenerative disease. In this application we propose a 5-year project aimed at this challenging bioengineering objective. The research will involve closely integrated studies employing molecular biology, organic synthesis, and biophysical/ electrophysiological analysis of prototype systems. A main focus of the proposed experiments is the GABAC receptor, a membrane receptor protein of retinal bipolar cells whose known properties make it well suited as a model system. Leading the research will be David R. Pepperberg, PhD, Principal Investigator (Ophthalmology and Visual Sciences, Univ. of Illinois at Chicago (UIC));and Co-Investigators Karol S. Bruzik, PhD (Medicinal Chemistry and Pharmacognosy, UIC), Tejal A. Desai, PhD (Biomedical Engineering, Boston Univ.), Jack H. Kaplan, PhD (Biochemistry and Molecular Genetics, UIC), Brian K. Kay, PhD (Biosciences Division, Argonne National Laboratory), Nalin M. Kumar, PhD (Ophthalmology and Visual Sciences, UIC), Guy C. Le Breton, PhD (Pharmacology, UIC), Jie Liang, PhD (Bioengineering, UIC), Haohua Qian, PhD (Ophthalmology and Visual Sciences, UIC), Sandra J. Rosenthal, PhD (Chemistry, Vanderbilt Univ.), and Robert F. Standaert, PhD (Chemistry, UIC). Lay language summary: The project's objective is to develop molecular structures that can restore vision in photoreceptor degenerative diseases such as age-related macular degeneration. These structures will be designed to attach to non-photoreceptor cells of the diseased retina that remain functional and to make these cells responsive to light.

ABSTRACT for EFRI-BSBA: Nanoactuation and Sensing of Neural Function for Engineering Future Biomimetic Retinal Implants and Therapies
PI: Laxman Saggere, Mechanical and Industrial Engineering, University of Illinois at Chicago (UIC)

Intellectual Merit

Retinal degenerative diseases such as age-related macular degeneration (AMD) affect over 10 million people in the US alone, causing a significant decline in the quality of their lives. Currently available therapies are at best only somewhat effective. Over the last two decades, several groups around the world have been pursuing the development of a retinal prosthesis, with the goal of providing a restorative aid for patients affected by retinal diseases due to photoreceptor degeneration. Nearly all of the current retinal prosthesis developments rely on the principle of stimulating the retina electrically, which is conceptually simple; however, a number of challenges still remain to be overcome in this approach and fully functional, long-lasting devices are not on the immediate horizon. On the other hand, a widely occurring mechanism of intercellular communication in the normally functioning retina as well as elsewhere in the nervous system is the chemical synapse. Inspired by the nature's complex mechanism of transducing visual information into chemical signals via the chemical synapse, the applicants envision an unconventional, but rational, approach to restore the lost functionality of photoreceptors: a light modulated chemical interface at the retina.

Toward this long-term vision of a chemically based retinal implant, the proposed project seeks to understand how the retina and retinal neurons respond physiologically to controlled focal presentation of chemical stimuli in vitro so that a general engineering framework for developing a prosthetic system based on the functionality of the diseased neurons can be further explored. There exist two distinct classes of chemicals, viz. native neurotransmitters and tethered synthetic biomolecules, that are promising as transmitters, and each offers certain unique advantages. Therefore, in this project, they propose to investigate the efficacy and feasibility of eliciting physiological responses of retinal neurons when focally stimulated by both types of chemicals delivered by means of specially engineered micro- and nanoscale delivery devices.

This novel approach is fundamentally different from the more common approach of electrically stimulating retinal neurons, and distinct from chemical-based strategies recently proposed by other groups. Thus, the main intellectual merit of this proposal lies in generating new scientific and technical knowledge that could be transformative to the development of a biomimetic retinal implant to restore lost or damaged retinal function. Ultimately, if successful, this research could lead to a new paradigm and breakthroughs in retinal prostheses.

Broader Impacts

The proposed project, if successful, could break new ground in the area of visual prosthesis and someday help provide vision perception to millions of people affected by retinal degenerative diseases. The devastating complications associated with vision loss, and the progressive aging of the US population with a corresponding increased incidence of AMD in otherwise healthy individuals, emphasize an urgent national need to develop effective prostheses and therapies for retinal degenerative diseases. Beyond the impact on vision health, this research could also lead to other novel drug delivery strategies and biomimetic therapies for treating a variety of neurological disorders such as Parkinson's.

The interdisciplinary collaboration of researchers with a diverse expertise in this project provides a unique opportunity and framework for interdisciplinary education and training of secondary school through postdoctoral students at the frontiers of engineering, neuroscience, and medicine. Four graduate students and one postdoctoral student will undertake interdisciplinary research addressing the tasks involved in this project in the investigators' labs across three different colleges at UIC. Several educational activities integrated with the proposed research including undergraduate research and outreach will be implemented.

DESCRIPTION (provided by applicant): Achieving the control, by light, of native voltage-gated sodium channels (NaVs) by a nanoscale molecular device could prove valuable as a vision repair therapy for photoreceptor degenerative diseases as well as for fundamental neuroscience research. The effectiveness of gold nanoparticles (Au NPs) in collecting and locally dissipating (as heat) the energy of visible light is well established. This, together with recent evidence that a sudden application of photothermal energy can promote NaV-mediated action potential generation, and the availability of small proteins possessing high specific affiniy for the NaV extracellular face, raise the possibility that a Au NP localized at the NaV ectomain by conjugation with the protein ligand affinity reagent can mediate selective photothermal NaV activation. The proposed exploratory project is aimed at testing the feasibility of this technolog. Aim 1 will provide the project's immediate foundation. Here we will determine the action of free Au NPs (i.e., not conjugated to protein ligand) on photo-induced transmembrane current in Xenopus oocytes, lipid bilayers, and isolated single ganglion cells of rat retina. The contributio of the Au NP to the light-elicited membrane current will be determined by comparing membrane currents elicited by photo-stimuli of similar intensity at wavelengths near to vs. distant from th nanoparticle's absorbance peak (~530 nm). The Aim 1 experiments will also establish experimental conditions needed for suitable timing/duration of the pulsed stimulating light required for this photothermal approach, and for aqueous solubilization of the Au NPs. In Aim 2 we will covalently couple the Au NP to the protein ligand Ts1, which has high affinity for the NaV domain II extracellular region. We hypothesize that close proximity of the Au NP to the membrane and its NaV target provided by attaching the Au NP to this NaV ligand will be key to minimizing the light level needed for NaV activation. We will construct Au NP-Ts1 conjugates in which a specific amino acid residue of the Ts1 is modified to serve as attachment site for the Au NP; we will determine the optimal structure of the conjugate by electrophysiological recording (experiments similar to those of Aim 1) and by analysis of cell-binding by fluorophore-tagged conjugate. Success in the project will establish feasibility of the investigated Au NP-based conjugate as a NaV photo-regulator. Specifically, it will encourage further development of this technology as a nano-prosthetic to enable NaV-mediated photo-signaling in ganglion cells of patients with advanced-stage photoreceptor degeneration. Leading the research will be David R. Pepperberg, PhD (Dept. of Ophthalmology and Visual Sciences, Univ. of Illinois at Chicago (UIC)); Francisco Bezanilla, PhD, and Stephen B. H. Kent, PhD (Depts. of Chemistry, Biochemistry and Molecular Biology, Univ. of Chicago); and Karol S. Bruzik, PhD (Dept. of Medicinal Chemistry and Pharmacognosy, UIC).

ABSTRACT Retinal ganglion cells (RGCs) convey visual signals to the brain in the form of action potentials, the initiation and axonal propagation of which depend on the activation of RGC voltage-gated sodium channels (NaVs). In photoreceptor degenerative diseases such as age-related macular degeneration (AMD), inner retinal neurons including RGCs in many cases remain intact and capable of generating action potential responses. RGCs thus represent a logical target for approaches aimed at restoring vision in advanced-stage AMD and related retinal diseases, by bypassing the nonfunctioning rod and cone photoreceptors and establishing direct RGC responsiveness to light. In a recent study of dorsal root ganglion cells (a non-retinal neuron widely studied as a model action-potential-generating cell type) and in hippocampal slice preparations, we have shown that gold nanoparticles (AuNPs) conjugated with a cell-targeting biomolecule enable robust light-induced NaV activation and resulting action potential generation. Essential features of the cell-targeted AuNP technique are: (i) functionalization of the light-absorbing AuNPs to localize them at or near the NaVs; (ii) upon the AuNP?s plasmon absorption of a millisecond/submillisecond light flash, AuNP radiation of the light energy as a nondamaging pulse of heat that creates a localized, transient, depolarizing capacitive current across the plasma membrane; and (iii) resulting activation, i.e., channel opening, of neighboring NaVs and thus action potential initiation by this depolarization. In this application we propose exploratory research to apply this AuNP approach to RGCs in the living eye of the rat. The project?s goal is to establish AuNP treatment conditions that achieve robust AuNP-mediated RGC photo-responsiveness in vivo. In rats for which rod and cone photoreceptor signaling to RGCs has been suppressed pharmacologically, we will intra-vitreally deliver AuNP conjugates designed for binding to the immediate vicinity of the RGC NaVs. Following treatment with the AuNP conjugates, we will employ in vivo recording of electroretinographic (ERG) signals associated with RGC activity, and of visual evoked potentials (VEPs), to analyze properties of RGC electrophysiological responses to AuNP photo-excitation. The research will involve variation of the size/structure of the AuNP, of the AuNP- conjugated RGC-targeting component, and of the duration and energy [(intensity) x (duration)] of AuNP- excitatory flashes. Accompanying the in vivo experiments will be all-optical stimulation/recording of AuNP- mediated action potentials in isolated rat retina, and histological analysis of retinas treated in vivo and in vitro with AuNPs. Results of the in vitro retina experiments will guide the selection of in vivo treatment conditions to be systematically investigated and facilitate interpretation of the in vivo data. Leading the research will be David R. Pepperberg, PhD (Univ. of Illinois at Chicago) and Francisco Bezanilla, PhD (Univ. of Chicago).