Molecular Mechanisms of Visual Signaling and Retinal Degeneration
Current staff (photo below Left to right):
Dr. Elena V. Olshevskaya, Senior Research Associate
Dr. Igor V. Peshenko, Instructor
Dr. Andrey B. Savchenko, Research Associate
Prof. Alexander M. Dizhoor, Martin and Florence Hafter Chair in
Pharmacology
Current collaborators: G. Fain lab (UCLA); C. Makino lab, T. Li lab (Harvard Medical School), R. Bush lab, P. Sieving lab (NIH), R. Barrett, Y.-S.Ho lab, Z.-H. Pan lab (WSU)
Current support: NIH, Lions of Pennsylvania, Pennsylvania Department of Health
Ongoing projects:
A. Mechanisms of signal transduction in photoreceptors.
Rods and cones respond to light by hyperpolarization of their plasma membrane, a process controlled by the level of cGMP in outer segments of photoreceptor. Guanylyl cyclase (retGC) produces cGMP in a calcium-sensitive manner, regulated by photoreceptor calcium-sensor proteins, GCAPs. My laboratory studies molecular mechanisms underlying activation of retGC by GCAPs and their role in signal transduction and retinal degeneration.
Dr. Igor Peshenko has demonstrated that regulation of GCAPs functions
as activators of retGC is a result of replacement of Ca2+ ions in their
metal-binding centers with Mg2+ ions, and he is now characterizing the role
of the individual Ca2+/Mg2+-biniding structures, EF-hands, in GCAPs in
activation and inhibition of retGC. [Reference]
B. Retinal degeneration caused by the abnormal cGMP synthesis
Mutations affecting synthesis of cGMP, the central signal transduction
substance in rods and cones, can result in blindness by causing
photoreceptor death. Retinal degeneration processes associated with either
lack of cGMP synthesis or its overproduction can be effectively mimicked in
mouse models that express mutant forms of retGC or GCAP. We developed
transgenic mouse models that carry mutations in GCAP1 found in human
patients with severe congenital retinal degenerations, and demonstrated that
a mutant form of GCAP elevates intracellular cGMP and calcium levels.
Immunocytochemical analysis of photoreceptor degeneration caused by the Y99C (top panel) mutant of GCAP1, done by Dr. Elena Olshevskaya. [Reference]
C. Rescue of photoreceptors from death caused by mutant GCAP1 using mutant rhodopsin that activates cGMP decay in the dark.
We introduced a mutant form of rhodopsin that activates cGMP hydrolysis in mouse rods in the dark into mouse rods affected by the Y99C mutant of GCAP1 that causes elevated cGMP synthesis in the dark. More than half of rods that would die due to the presence of the Y99C GCAP1 remain alive after many months, rescued by the presence of mutant rhodopsin.
Loss of retinal electrical responses to light and their rescue by mutant rhodopsin recorded using dark-adapted electroretinography (ERG) by Dr. Andrey Savchenko ARVO2006 abst. 1093, [Reference]
D. “Prosthetic” photosensitive neurons for testing new approaches to gene therapy of complete blindness.
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Degeneration of rods and cones can completely deprive retina of
photosensitive neurons required for visual signal to occur. We have
produced an algae light-sensitive ion channel, CHOP2 , in a form that allows
to visualize its presence in the cells and expressed the channel in a
functional form, first in HEK293 and then in retinal ganglion cells (in
collaboration with Dr. Z.H. Pan, WSU), which evoked visual cortical
responses in mice lacking photoreceptors. [Reference]
SELECTED RECENT PUBLICATIONS
Dizhoor, A. Site-directed and natural mutations in studying functional domains in guanylyl cyclase activating proteins (GCAPs). Adv Exp Med Biol. 514:291-301 (2002).
Peshenko, I.V., Dizhoor, A.M. Guanylyl Cyclase Activating Proteins (GCAPs) Are Ca2+/Mg2+-Sensors: Implications for Photoreceptor Guanylyl Cyclase (RetGC) Regulation in vitro and in vivo. J Biol Chem. 279 16903-16906 (2004)
Olshevskaya, E.V., Calvert P.D., Woodruff, M.L., Peshenko I.V., Savchenko A.B.., Makino C.L., Ho Y-S., Fain G.L., Dizhoor, A.M. The Y99C mutation in guanylyl cyclase activating protein 1 (GCAP1) increases intracellular Ca2+ and causes photoreceptor degeneration in transgenic mice. J. Neuroscience 24, 6078-6085 (2004)
Peshenko, I.V., Moyiseev, G.P, Olshevskaya, E.V., Dizhoor, A.M. Factors that determine Ca2+- sensitivity of photoreceptor guanylyl cyclase. Kinetic Analysis of the Interaction between the Ca2+-Bound and the Ca2+-Free Guanylyl Cyclase Activating Proteins (GCAPs) and Recombinant Photoreceptor Guanylyl Cyclase 1 (RetGC-1). Biochemistry 43, 13796-13804 (2004)
Peshenko, I.V., Olshevskaya, E.V., Dizhoor, A.M. Ca2+-dependent conformational changes in guanylyl cyclase activating protein 2 (GCAP-2) revealed by site-specific phosphorylation and partial proteolysis. J. Biol. Chem. 279, 50342-50349 (2004)
Dizhoor A.M., Olshevskaya E.V., Peshenko I.V. (2005) Calcium sensitivity of photoreceptor guanylyl cyclase (RetGC) and congenital photoreceptor degeneration: modeling in vitro and in vivo. In: "Neuronal Calcium Sensor Proteins” (Philippov, P.P. & Koch, K-W, eds.), Nova Science Publishers, Inc, NY. In Press.
Makino, C.L., Wen X.H., Michaud N., Peshenko I.V., Pawlyk B., Brush R.S., Solovyeva M., Liu X., Woodruff M.L., Calvert P.D., Savchenko A.B., Anderson R.E., Fain G.L., Li T., Sandberg M.A., Dizhoor A.M. Effects of Low AIPL1 Expression on Phototransduction in Rods. (2006) Investigative Ophthalmology and Visual Sciences, in press
Bi, A., Cui, J., Ma, Y.-P., Olshevskaya, E., Pu, M., Dizhoor, A.M., Pan, Z.-H. (2006) Ectopic Expression of a microbial-type rhodopsin restores visual response in mice with photoreceptor degeneration. Neuron, 50, 1-11