Robert Molday

Rod and cone photoceptors are specialized neurons which lie adjacent to the retinal pigment epithelium (RPE) cell layer at the back of the retina. Rod cells are involved in vision under low light intensity and cone cells are involved in vision under normal lighting conditions, color vision, and high visual acuity. These cells function in phototransduction, the process in which light is detected and converted into electrical signals as the primary event in vision. Phototransduction takes place in a specialized compartment known as the outer segment. This compartment is connected to the rest of the cell through a thin cilium.  Photoreceptor cells together with RPE cells are also involved in the regeneration of 11-cis retinal for the regeneration of opsins via a series of enzyme-catalyzed reactions known as the visual cycle. Our current research is focused on 1) determining the molecular structure and function of key photoreceptor proteins involved in phototransduction, the visual cycle, outer segment structure, morphogenesis, and renewal and 2) elucidating the role of various proteins in retinal degenerative diseases that are a major cause of blindness in the world population.  We are using this information to develop novel gene and drug-based therapies as promising treatments for these diseases. Several proteins that we are now studying are ABCA4, an ABC transporter linked to Stargardt disease, retinoschisin, an extracellular protein involved in retinal cell adhesion and associated with X-linked retinoschisis, guanylate cyclase, a key enzyme involved in the synthesis of cGMP and associated with Leber Congenital Amaurosis, peripherin-2, a structural protein responsible for disc rim curvature and associated with retinitis pigmentosa and macular dystrophy, RD3 involved in guanylate cyclase trafficking, and P4-ATPases linked severe human diseases.

Phototransduction: Light isomerizes 11-cis retinal to all-trans retinal in rhodopsin. This activates the G-protein, (transducing), which in turn activates phosphodiesterase (PDE) resulting in a decrease in cGMP.  This results in the closure of the cGMP-gated channels to the influx of cations (Na, Ca) and a hyperpolarization of the cell. 

Diagram showing the distribution of key proteins in the disk and plasma membrane of the rod outer segment.  Rhodopsin is present in the flat part of the disk membrane and in the plasma membrane. Peripherin-2/rom-1 (Per2/Rom1) complex along with ABCA4 and guanylate cyclase is present along the rim region of disk membranes. The cGMP-gated channel (CNG) consisting of A1 and B1 subunits is present in the plasma membrane and associates with the Na/Ca-K exchanger (NCKX1). The Per2/Rom1 appears to interact with the GARP domain of the CNG channel B1 subunit.

Structure of the ATP-Binding Cassette (ABC) transporter ABCA4 in the open state containing the bound substrate N-retinylidene-phosphatidylethanolamine (NRPE) and the closed state containing ATP bound to each nucleotide-binding domain.

Rod and Cone Photoreceptor Cells:

The outer segment is a specialized compartment of rod and cone photoreceptor cells where light is captured and converted into an electrical signal in a signaling process known as phototransduction. The rod outer segment consists of a stack of over 1000 discs surrounded by a plasma membrane. We are identifying and characterizing novel membrane and cytoskeletal proteins important in phototransduction, generation and renewal of outer segments, protein trafficking from the inner to the outer segment, and photoreceptor cell survival.

ABCA4 and Stargardt Macular Degeneration:

ABCA4 is a member of the family of ATP-binding cassette (ABC) transporters that resides in outer segment disc membranes. Over 800 mutations in ABCA4 are responsible for a relatively common early onset retinal degenerative disease known as Stargardt macular degeneration. We have recently shown that ABCA4 functions as retinoid-lipid transporter which plays a key role in the removal of potentially toxic retinoids from photoreceptors. We are currently studying the structure, function and mechanism of transport of ABCA4 and disease-associated mutants in order to determine how selected mutations in ABCA4 cause Stargardt disease. We are also exploring the use of drug and gene therapy as potential treatments for Stargardt disease.

RD3, GC, and Leber Congenital Amaurosis:

RD3 is a 23 kDa protein encoded by a gene associated with Leber Congenital Amaurosis Type 12 (LCA12), a inherited retinal dystrophy which causes severe vision loss in infants. Mutations in RD3 also cause rod and cone photoreceptor degeneration in the rd3 mouse and rcd2 collie which serve as valuable animal models for LCA12. We have recently shown that RD3 plays a crucial role in the trafficking of guanylate cyclase (GC) to rod and cone outer segments. GC is a key protein involved in the production of cGMP, the second messenger for phototransduction, and associated with Leber Congenital Amaurosis Type 1 (LCA1). Ongoing studies are directed toward elucidating the mechanism by which RD3 facilitates vesicle trafficking of GC to outer segments. We are also carrying out studies to determine if the delivery of the Rd3 gene to photoreceptors using AAV vectors can restore vision and maintain photoreceptor cell survival in the rd3 mouse as a proof-of-concept for the application of gene therapy as a possible treatment for LCA12.

Phospholipid Transport across Membranes:

P4-ATPases and ABCA Transporters function in the transport of phospholipids to generate lipid asymmetry across biological membranes and facilitate lipid homeostasis. We have recently purified and characterized a P4-ATPase known as ATP8A2 and have shown that it is localized in rod and cone photoreceptors where it transports or flips phosphatidylserine and phosphatidylethanolamine across cell membranes, a process that is important in vesicle trafficking and phototransduction. We have also shown that both ABCA4 and ABCA1 function as phospholipid transporters, but transport phospholipids in opposite directions. Current research is focused on analysis of the structure and function of various P4-ATPases and ABCA transporters in order to define in detail their mechanism of action and role in biological processes and disease.