Genetics in the Retina
Genetics of Cell Differentiation and Survival of Retinal Neurons (Gan)
The mammalian retina is a major target of genetic diseases that cause blindness due to the degeneration of retinal neurons. In order to understand the disease processes, it is very crucial to elucidate the fundamental mechanisms regulating the normal development and maintenance of these neurons at the molecular level. Dr. Lin Gan's research is centered on identifying the genes required for neuron differentiation and survival, investigating the genetic pathways involved in these processes, and developing therapies for blindness via gene therapy and stem cell replacement. He is currently investigating the roles of three classes of transcription factors (TFs), the basic helix-loop-helix (bHLH), POU-homeodomain (POU-HD), and LIM-domain TFs, in the formation and maintenance of mouse retina. Using homologous recombination in mouse embryonic stem (ES) cells to mutate these TF genes, he has shown that these TFs function in a cascade to regulate the differentiation of neuronal progenitor cells into specific types of neurons and to regulate the maturation and survival of post-differentiation neurons.
Restoration of Vision to Blinded Retina by Light-Gated Channels (Merigan)
Channelrhodopsin2 (ChR2) expressed in inner retinal neurons can render them photosensitive and has been shown to restore light-driven behavior in experimental animals that are blind due to retinal degeneration. These encouraging results suggest the prospect that ChR2, or a related optogenetic approach, may someday restore vision in blind humans. However a prerequisite to a human prosthetic based on this approach is the study of the perceptual abilities that ChR2 can provide; acuity, contrast sensitivity, shape perception, and motion and orientation discrimination. The optogenetic prosthesis will be created by expressing ChR2 in retinal ganglion cells (RGCs) that have lost photoreceptor input at a small retinal location. Dr. William Merigan examines the efficacy of the prosthetic with two methods: the light responses of individual ganglion cells recorded in vivo with adaptive optics imaging of a fluorescent, genetically-encoded, calcium indicator, and behavioral testing of visual thresholds to quantify the extent to which perceptual abilities can be restored, relating ChR2-mediated visual performance to the restored receptive field properties of individual RGCs at the same retinal location. Once ChR2 is shown to successfully drive ganglion cells, these methods can be used to quantify basic parameters about the performance of the prosthetic including sensitivity, dynamic range, and spatial information capacity.
In Vivo Adaptive Optics Imaging of the Retina
Calcium Imaging of Retinal Ganglion Cell Function (Merigan)
Dr. William Merigan, in collaboration with Drs. David Williams and Lu Yin, has developed a novel method for in vivo imaging of the light response of retinal ganglion cells. Ganglion cells are challenging to study because they are transparent and cannot be imaged with reflectance or single-photon fluorescence methods. The method Merigan developed uses insertion of a fluorescent calcium indicator, G-CaMP, into ganglion cells by a viral vector often used in gene therapy, followed by adaptive optics imaging of the increased fluorescence that results from visual activation. This new technique offers several advantages over existing methods for retinal physiology. Since it is an in-vivo method it can track the physiological response of single cells over weeks to months, making it valuable for extended studies of disease development or therapeutic intervention. It simultaneously images the physiological response of all G-CaMP expressing cells within the imaging field, providing a direct comparison of the response of numerous types of cells.
Application of Novel Imaging Methodologies to the Retina (Hunter)
Dr. Jennifer Hunter is developing new imaging modalities for studying human retina to extend the capabilities of imaging, which presently uses reflectance and single photon fluorescence methods. She is working to bring multi-photon imaging of the retina to the clinic because of the great advance this would bring for imaging the function (and dysfunction in eye disease) of most retinal neurons. Multi-photon imaging uses a short-pulse (femtosecond) laser to produce a fluorescent signal by the absorbtion of multiple photons by a fluorophore. Some of the most important intrinsic retinal fluorophores such as flavin adenine dinucleotide (FAD) and reduced nicotinamide adenine dinucleotide phosphate (NADPH) cannot be examined by existing single-photon fluorescent methods, since wavelengths required to activate these fluorophores cannot penetrate the eye’s optics. However, 2-photon imaging can drive these fluorophores with longer wavelengths of approximately 700 nm, which easily penetrate the optics to the retina. The 2-photon method is also advantageous even for fluoropohores that can be activated through the optics of the eye since long wavelengths are much less damaging to the retina than short wavelengths and long wavelengths minimally activate retinal photoreceptors making it possible to activate fluorescence without stimulating vision. The photo above shows in vivo 2-photon images of cone photoreceptors in primate retina.
Imaging Studies of the Progression of Retinal Eye Disease (Chung)
Dr. Mina Chung is studying disease progression across the retina using adaptive optics retinal imaging methodologies developed at Rochester. She combines these methods with genetic screens, and traditional and advanced imaging methods such as optical coherence tomography (OCT) and fundus autofluorescence (AF) imaging. The photo below shows high-resolution images of individual cones superimposed on a low resolution fundus image of the same retina.
Her studies are aimed at understanding mechanisms of retinal disease and evaluating new therapeutic interventions and she uses reflection imaging of rod and cone photoreceptors and single-photon fluorescence imaging of retinal pigment epithelium (RPE). Dr. Chung’s studies are primarily focused on age-related macular degeneration (AMD), macular telangiectasia, and geographic atrophy.
Photoreceptors and Retinal Pigment Epithelium (RPE) (Hunter)
Dr. Hunter, in collaboration with Dr. David Williams, uses single-photon fluorescence of retinal pigment epithelium to image these cells, which are located near the base of the retina, next to rod and cone photoreceptors, and which are involved in many retinal diseases. this slide shows a fluorescence image of the mosaic of RPE cells in and around primate fovea. RPE cells play a prominent role in the health of photoreceptors and the trafficking of photopigments involved in the retinoid cycle.
Vulnerability of Retinal Neurons to Light Damage (Hunter, Merigan & Williams)
The exquisite responsivity of the retina to light makes it also very sensitive to light damage. Light exposure limits published by the American National Standards Institute (ANSI) committee, of which Dr. Jennifer Hunter is a member, are designed to protect people from excessive light, but some parameters of light exposure have not been studied well enough to ensure that retinas are not adversely affected. For example, Dr. Hunter and colleagues discovered that ANSI standards did not adequately protect people from long-term exposure to low levels of light, conditions used in adaptive optics imaging of fluorescence. She is currently studying such long-term exposures to determine the impact of the wavelength of the light on this phototoxicity. She is also examining the potential for retinal damage of the novel imaging methodologies she is developing, for example two-photon imaging.