Clegg Lab

How does something as complex, beautiful, and functional as an eye develop? How do neurons become precisely aligned and connected in intricate networks that allow vision and thought? And what goes awry in the case of neurodegenerative diseases? Finally, can stem cells be applied in novel therapies to treat such diseases, especially Age-related Macular Degeneration? These are the principal questions that drive our current research in the areas of neural development and neural disease.

Neural Development

We are interested in identifying and understanding the external molecular cues that guide the development of neurons. These include secreted extracellular matrix (ECM) molecules and cell surface molecules that influence cell fate, cell adhesion and migration, axon pathfinding, and synapse formation. All of these events are mediated by specific neuronal receptors that recognize extracellular molecules and transduce signals across the membrane to elicit intracellular changes. By employing a broad arsenal of modern molecular techniques, we hope to gain an understanding of how neurons respond to these factors and what role these interactions play in vivo. One research interest has been the functions of integrin receptors in the developing nervous system, focusing on retinal neurons and sympathetic neurons that innervate the heart. Integrins are transmembrane receptors for ECM proteins such as laminin and fibronectin, and cell surface adhesion molecules such as VCAM-1. Most recent studies have centered on the α4β1 integrin. While this integrin has been shown to play important roles in inflammatory responses, we were the first to show it is also expressed in the nervous system. In the retina, we found that this receptor is important in neuronal survival. In the heart, we showed that the α4β1 integrin is important in the elaboration of sympathetic axons into cardiac tissue (see right figure).

We have also investigated a number of the ECM components found in the nervous system, such as laminins, fibronectins, collagens, and the matrix metalloproteinases that degrade them. We have found novel roles for the ECM proteins osteopontin (see left figure) and thrombospondin-4 in the developing retina. Both molecules reside in the optic nerve at the time retinal ganglion cells (RGCs) extend axons to the brain, and we have shown that purified osteopontin supports RGC axon growth in vitro. The integrin α4β1 on RGCs serves as an osteopontin receptor that mediates axon extension on osteopontin. Thrombospondin-4 may play a role as an organizer of optic nerve ECM. Further experiments are underway using genetic strategies in mice to investigate the functions of these ECM proteins in the eye.

Neural Disease

One of the reasons for studying development is this: perhaps knowledge gained would be useful in designing treatments for degenerative diseases. If we could kick start the developmental program in a degenerating retina, perhaps we could alleviate disease. Recently, we have begun collaborating with the Center for the Study of Macular Degeneration (CSMD) within the Neuroscience Research Institute at UCSB, with the intent of establishing a second research emphasis in retinal disease.

Loss of vision in Age-Related Macular Degeneration (AMD), a leading cause of blindness in this country, is a result of degeneration of rods and cones in the macular region of the central retina, which is responsible for high acuity vision. Death of the photoreceptors appears to be a consequence of degeneration of neighboring RPE cells. The disease is poorly understood and there is no good treatment or cure.

The formation of drusen, abnormal deposits in the ECM, is an important hallmark of AMD. Typically, drusen lie between the RPE basement membrane and the inner collagenous layer of Bruch's membrane and contain a variety of ECM and molecules. Some investigators have hypothesized that drusen may result from the failure to dispose of RPE-derived molecules (including ECM), or may be due in part to an inflammatory immune reaction. Drusen formation appears to precede the occurrence of visual symptoms in AMD eyes and is hypothesized to be a cause of the disease. The molecular events that underlie drusen formation are not well established.

Our interest in AMD was sparked by the apparent involvement of the ECM in disease progression. Mutations in the Tissue Inhibitor of Metalloproteinase-3 (TIMP-3) have been linked to Sorsby's Fundus Dystrophy, a blinding eye disease with some similarities to AMD. We investigated the distribution and activity of matrix metalloproteinases (MMPs) and TIMPS in human RPE-choroid from donor eyes with and without a clinical history of AMD. We found that TIMP-3 is concentrated in drusen, and that drusen are coldspots for proteolysis activity (see left figure).

In addition, mutations in a family of ECM proteins called fibulins have recently been shown to give rise to AMD. We have initiated studies of the basic biology of fibulin-5 and fibulin-6, and have shown that these are expressed by RPE and are components of drusen. Future experiments will examine fibulin functions as well as expression of fibulins in human RPE-choroid from donor eyes with and without a clinical history of AMD.

We are also investigating the role of ECM proteins in other eye conditions. We have recently found evidence that osteopontin contributes to disease progression in several mouse models of eye disease by recruiting immune cells to damaged areas. Models under study include experimental immune uveitis (an autoimmune disease) and glaucoma. The right figure shows results from an experiment where purified recombinant osteopontin has been injected into a mouse eye, resulting in retinal folding and influx of immune cells. New experiments indicate a role for ostepontin in a mouse model of exitotoxcity related to glaucoma.

Stem Cells

Embryonic, induced pluripotent (iPS), and adult stem cells have great potential for treating a variety of diseases, including eye maladies such as macular degeneration. We have begun studies aimed at understanding the basic biology of how stem cells differentiate into ocular cells, especially RPE cells. The right figure shows RPE cells derived from human embryonic stem cells, which are similar but not identical to native RPE. We have recently shown that RPE differentiated from iPS cells can rescue vision in a rat model of retinal dystrophy. We are working with a "Disease Team" of investigators from USC, Caltech, City of Hope, University College, London, and Geron, Inc, ranging from basic to clinical, to translate this work to the clinic. In another project, adult human adipose stem cells are being investigated, which might have applications in soft tissue regeneration. Interdisciplinary collaborations with materials scientists to engineer synthetic matrix for stem cell growth and differentiation are also underway. Novel, bio-mimetic substrates are being designed to stimulate stem cell integrin receptors that signal to bring about cell survival, proliferation, or differentiation.