Shu-Zhen Wang - US grants

DESCRIPTION (provided by applicant): The long-term goal of this project is to elucidate the molecular regulation of photoreceptor production in the vertebrate retina. This knowledge is imperative to the development of effective stem cell-based photoreceptor replacement therapies. Much needs to be learned about the identities of the genes involved and how they contribute to the selection of the photoreceptor fate from among the other options. Studies during the previous funding period identified neuroD as an instrumental player in photoreceptor differentiation, in a transcriptional pathway of ngn2->neuroD->RaxL. In this pathway, ngn2 functions in multipotent progenitors and its downstream genetic targets include neuroD;neuroD elicits a photoreceptor development program, including the expression of homeobox gene RaxL. In this application we will test the hypothesis that photoreceptor production employs ngn1 at a key step between ngn2 and neuroD. Aim 1 examines whether ngn1 is expressed at the right place and the right time to be a major gene in leading progenitors to the photoreceptor path. Aim 2 investigates whether ngn1 expressly steers progenitors to the photoreceptor path and, thus, leads exclusively to photoreceptor production. Aim 3 determines whether ngn1 is required for photoreceptor production. Aim 4 addresses how ngn1 genetically relates to ngn2 and neuroD during photoreceptor production. These studies will be carried out with a battery of techniques from molecular biology, cell biology, developmental biology, and genetics. This project promises to shed light on the transcriptional regulation governing photoreceptor production. Furthermore, it bears clinical implications. The identification of key genetic players in photoreceptor production will capacitate efficient in vitro or in vivo photoreceptor generation for studies with therapeutic goals. These studies are timely in this era of heightened interest in stem cell research for replacement therapies.

DESCRIPTION (provided by applicant): The overall objective of this research is to understand the neuronal circuitry that processes information through the inner plexiform layer (IPL) of the mammalian retina. Because our knowledge of cone bipolar cell connectivity is insufficient, as is the knowledge of rabbit ganglion cell synaptology, the experimental design is focused on specific cone bipolar cell circuits, the excitatory input they receive from cone pedicles and the excitatory input they exert onto ganglion cell dendrites Therefore, our network analysis involves exploiting the cytoarchitectural organization of the retina, which is the keystone to understanding retinal function by examining the connectivity of identified cone bipolar cell axon terminals that excite ON-OFF directional selective (DS) and OFF-alpha ganglion cells. We have described a lateral feed-forward inhibitory microcircuit involving two different bipolar cells; the dyad of one bipolar cell directly excites the DS cell, while the dyad of the other excites a starburst amacrine cell which feeds forward to synapse with the DS cell. lmmunolabeling and intracellular staining, in combination with confocal microscopy, are used to identify bipolar cells synapsing with the DS cell in order to focus the electron microscopy (EM) analysis. After the bipolar cells are identified, the axon terminals of these intracellular!y stained cells are serially reconstructed with EM to examine their synaptology; two constrasting electron dense markers, pro embedding gold-substituted silver intensified peroxidase and DAB/peroxidase, are used to establish pro- and postsynaptic interactions between two cells. Combined microwave and chemical fixation and/or rapid-freezing are used with various pro- and postembedding EM immunolabeling techniques involving DAB/peroxidase and immunogold to identify the GABA and cholinergic synapses and determine how these receptors are used by the starburst amacrine cell to communicate with the DS cell and each other. Whole cell recordings are used to characterize the ionotropic glutamate receptors on the dendrites of OFF-bipolar cell(s) synapsing with OFF-alpha ganglion cells and to determine whether the bipolar cell(s) primarily uses the same glutamate receptor to synapse with the OFF-ganglion cell. Once the functional glutamate receptors are characterized, double immunolabeling is used to confirm their presence on the dendrites of the ganglion cell where it receives excitation from dyad synapses.

The long-term goal of this project is to produce new photoreceptors for cell replacement. Photoreceptor replacement holds great promise in treating visual impairments caused by photoreceptor degeneration. At the same time, it presents the need for a supply of differentiating photoreceptors, because the human neural retina lacks regeneration capability. To address this critical barrier in developing photoreceptor-replacement therapies, we take a rather unconventional approach to generate differentiating photoreceptors - reprogramming RPE cells with a pro-photoreceptor gene to channel RPE's well-known capabilities of proliferation and plasticity towards photoreceptor production. Studies with chick cells raise the exciting possibility of deriving new photoreceptors from the RPE through gene-directed reprogramming. Interesting as it stands, it is time to test the hypothesis that mammalian RPE cells can be reprogrammed to give rise to photoreceptor cells. Validation of the hypothesis bears clinical and societal significance. To test the hypothesis, we designed two sets of complementary studies. The first set directly examines cultured human RPE cells for their capacity to produce photoreceptor cells under the guidance of a pro-photoreceptor gene ngn1. Human RPE cells will be virally transduced with ngn1 to initiate photoreceptor differentiation. The cell culture will then be analyzed for de novo production of photoreceptor-like neurons at the levels of gene expression, cellular morphology, and functional physiology, in vitro and in vivo after transplantation into the eyes. A direct test with human cells bears high relevance to the development of potential therapy. The second set investigates whether new photoreceptor cells will be generated from the mouse RPE ectopically expressing pro-photoreceptor gene ngn1. Ectopic ngn1 expression in the RPE will be achieved using viral delivery and transgenics. The ngn1-RPE will then be subjected to conditions, such as the in vivo environment of photoreceptor degeneration, that may unleash the experimental RPE's potential to give rise to photoreceptor cells. This will be followed by analyses for de novo generation of photoreceptor cells at molecular, cellular, and physiological levels. In addition to testing our hypothesis, a demonstration of RPE -> photoreceptor reprogramming in mice will provide scientific evidence for future investigation into RPE as a convenient source of photoreceptors for in situ cell replacement without cell transplantation. Together, the studies promise information vital to using the RPE to repopulate the retina afflicted with photoreceptor degeneration.

PROJECT SUMMARY/ABSTRACT One of the current challenges in restoring vision through regeneration of the retina is the lack of means to activate endogenous progenitor cells for the production of a sufficient number of functional photoreceptors, as reflected in the NEI Audacious Goal Initiatives ( . To undertake this challenge, this project investigates a rather unconventional approach to photoreceptor regeneration ? tapping into the regenerative potential of a nearby tissue, the retinal pigment epithelium (RPE), for photoreceptor regeneration in the mouse eye using gene-directed reprogramming. Our long-term goal is to elicit photoreceptor regeneration in the mammalian eye for replacement without cell transplantation. NOT-EY-14-003) Recent studies using mammalian RPE cell cultures and transgenic mice have produced evidence for the feasibility of this unconventional approach to photoreceptor regeneration in the eye. Moving forward, this project tests the hypothesis that mammalian RPE's regenerative potential, when unlocked by proneural gene neurogenin3 (or neurogenin1), can produce functional photoreceptors and new RPE cells to sustain RPE structure and function. Experiments are designed to achieve two specific aims. Specific Aim 1 determines whether an RPE cell, when primed by neurogenin3, directly undergoes RPE-to-photoreceptor transformation (i.e., transdifferentiation), or if it first goes through a transformation into a progenitor-like stage generating cells with the potentials to differentiate into photoreceptor cells and RPE cells. This study promises to shed light on the cellular mechanisms of how the RPE gives rise to photoreceptor cells and replenishes itself. Specific Aim 2 determines whether functional photoreceptors can be generated in adult mouse using a gene delivery approach compatible with human application, and whether the RPE will be preserved structurally and functionally. This study is expected to produce compelling evidence supporting human feasibility of engaging the RPE as a convenient source of new photoreceptors for functional repair in situ without involving cell transplantation and the associated risks and potential complications.