early one in 80 persons is a carrier, usually unknowingly, of retinitis pigmentosa, or RP, a group of retinal degenerative disorders that destroys the eye's photoreceptors and eventually causes blindness. Unraveling the insidious disease's riddle is of crucial importance, since RP is considered a model for other genetically transmitted conditions. Debora B. Farber, professor of ophthalmology and associate director of the Jules Stein Eye Institute, and her group have already identified one culprit, a gene that encodes a component of the phototransduction cascade essential to vision. When a photon of light is absorbed by visual pigment, it activates rhodopsin (R*), which in turn activates transducin (T), a protein formed by a (Ta), b (Tb) and g (Tg) subunits. Subsequently, Ta separates from Tb and Tg, activating cyclic guanosine monophosphate-phosphodiesteras e (cGMP-PDE), an enzyme made of four subunits. a-PDE and b-PDE both have catalytic activity; the two g-PDEs are inhibitory, thus regulating overall cGMP-PDE activity. The enzyme then breaks down cGMP, which gates the channels of the photoreceptor cell plasma membrane and keeps them open in the dark. The reduction of cGMP causes the channels to close, generating an electrical signal to the brain to relay the message that light has been seen by the eye.
Farber initially isolated the gene that causes retinal degeneration in the rd mouse, a rodent which inherits a blinding disease similar to human RP. This gene encodes the beta subunit of cGMP-PDE (b-PDE). Without normal b-PDE, the cGMP-PDE complex cannot be formed and cGMP-PDE activity in the retina dwindles. As rd mice cannot degrade cGMP, toxic levels of cGMP build up, leading to photoreceptor death and ultimately to blindness. Using transgenic techniques—prenatally replacing the abnormal b-PDE gene in the rd mouse with a normal counterpart—Farber and colleagues from Caltech and the University of Chicago produced mice free of disease, confirming that the b-PDE gene defect causes retinal degeneration in the rd mouse.
Only five or six percent of patients with autosomal recessive RP seem to have mutations of the b-PDE gene, but these mutations are the most common yet found in autosomal recessive RP, which constitutes 33 percent of all cases of RP (autosomal dominant represent some 20 percent; x-linked, 10 percent; and simplex, of unknown origin, the balance). "This gene is very susceptible to mutations," notes Farber.
"changes in even one nucleotide are common; each mutation causes a different problem in the resulting protein."
In the rd mouse, photoreceptor degeneration begins postnatally around day nine, with blindness occurring in four weeks. In humans with RP, onset can occur anytime between infancy and adulthood and may progress over months or decades.
In addition to screening the DNA of human families with autosomal recessive RP for mutations in the b-PDE gene, Farber's group is investigating the involvement of transcription factors which may enhance or repress the expression of the b-PDE gene. The researchers are using biochemistry, cell and molecular biology and genetics to examine the roles of the genes and proteins in inherited retinal degenerations.
Recently, work on "g-PDE knockout mice," mice in which the gene encoding g-PDE had been deleted from their genome, brought surprising results. Without inhibitory g-PDE, the activity of cGMP-PDE should be very high. "But instead of very low levels of retinal GMP caused by a constantly active enzyme," says Farber, "mice without the g-PDE had cGMP-PDE that wasn't working at all—a retinal degeneration phenotype similar to that of the rd mouse. Therefore, g-PDE is not only necessary to regulate cGMP-PDE activity, it is also important for assembly of the alpha, beta and gamma subunits into a complex."
Since the absence of g-PDE can cause retinal degeneration in mice, it may play a role in retinal degeneration in humans. Delineating the specific functions of the genes and proteins required for photoreception could lead to the development of treatments for RP, cone dystrophies and possibly other retinal disorders.
In utero replacement of deficient genes (similar to the production of transgenic mice) would be complicated in humans, so Farber has been developing gene therapy approaches "that eventually could be offered to RP-afflicted people at a young age." Researchers in her lab have already introduced the normal b-PDE gene into a modified adenovirus that serves as a shuttle, and have injected this construct subretinally into the eyes of rd mice.
Their results have been very promising, since prolonged expression of b-PDE and rescue of rod photoreceptor cells were achieved for at least three months in the rd mouse retina. As for Farber, she'll be happy "to help at least one person regain vision." —Sue Larson
work to identify
a bedeviling recessive
blindness could lead
to an early cure