The mechanisms triggering photoreceptor cell death remain poorly understood, hindering the development of effective therapies for retinal degenerations affecting more than 1.5 million people. The long-term goal of this study is to elucidate molecular signals that trigger photoreceptor cell death. Recently, we identified a subclass of degenerations in which persistent activation of phototransduction signaling caused rod cell death. We hypothesize that cGMP hydrolysis resulting from persistent transducin signaling causes retinal degeneration. The drop in cGMP increases the fraction of rod cyclic nucleotide gated (CNG) channels in the closed configuration, reducing calcium entry into photoreceptors. Calcium depletion is one possible cause of degeneration. However, low intracellular calcium can also stimulate guanylate cyclase activity to synthesize cGMP. Depending on the relative rates of cGMP hydrolysis and synthesis, this may result in a futile cycle that leads to either low or elevated cGMP levels, closing or opening channels to cause insufficient or excess calcium, respectively. To test this hypothesis, we will create transgenic mice with mutations affecting genes that modulate calcium influx and efflux in rod cells, namely the rod CNG a-subunit and the rod Na+, Ca2+/K+ exchanger. Our goal is to examine whether perturbations in cGMP levels, which alter intracellular calcium, is a mechanistic cause of retinal degenerations.
Specific Aim 1. To test the hypothesis that a mutation that predominantly sets the rod cGMP nucleotide-gated channel in the closed configuration will induce degeneration much the same as constitutive activation of the phototransduction cascade will. We will introduce an R553Q CNGA1 knock-in mutation in transgenic mice that reduces cGMP binding affinity 30-fold, closing a larger fraction of CNG channels. The effect on retinal morphology, cGMP and calcium levels will be assessed.
Specific Aim 2. To test the hypothesis that retinal degeneration associated with elevated cGMP in the """"""""rd"""""""" mouse retina is caused by excessive Ca2+ in rod outer segments and can be ameliorated by reducing intracellular Ca2+. We will genetically reduce intracellular calcium in the """"""""rd"""""""" mouse by over expressing the Na+,Ca2+/K+ exchanger (NCKX1) expressed from the rod opsin promoter, thus increasing efflux of calcium. If the hypothesis is correct, degeneration in rd, NCKX1 double mutant mice will be slowed compared to the rate of degeneration in rd mice.
Specific Aim 3. To test the hypothesis that mutations in the NCKX1 gene, which regulates Ca2 efflux from rod outer segments, cause retinal degeneration by increasing intracellular calcium. An I906T rod Na+,Ca2+/K+ exchanger (NCKX1) knock-in mutant mouse will be created. In vitro studies show reduced transport activity by this mutant. The reduced calcium efflux is predicted to cause a rise in intracellular calcium. If elevated calcium is a cause of retinal degeneration, we predict that degeneration will occur. The proposed studies will elucidate the physiological relationship and role of cGMP and Ca2+ in retinal disease and normal photoreceptor function.
|Makino, Clint L; Wen, Xiao-Hong; Michaud, Norman A et al. (2012) Rhodopsin expression level affects rod outer segment morphology and photoresponse kinetics. PLoS One 7:e37832|
|Maeda, Akiko; Okano, Kiichiro; Park, Paul S-H et al. (2010) Palmitoylation stabilizes unliganded rod opsin. Proc Natl Acad Sci U S A 107:8428-33|
|Pang, Ji-Jie; Gao, Fan; Lem, Janis et al. (2010) Direct rod input to cone BCs and direct cone input to rod BCs challenge the traditional view of mammalian BC circuitry. Proc Natl Acad Sci U S A 107:395-400|
|Park, Paul S-H; Sapra, K Tanuj; Jastrzebska, Beata et al. (2009) Modulation of molecular interactions and function by rhodopsin palmitylation. Biochemistry 48:4294-304|
|Malanson, Katherine M; Lem, Janis (2009) Rhodopsin-mediated retinitis pigmentosa. Prog Mol Biol Transl Sci 88:1-31|
|Wen, Xiao-Hong; Shen, Lixin; Brush, Richard S et al. (2009) Overexpression of rhodopsin alters the structure and photoresponse of rod photoreceptors. Biophys J 96:939-50|
|Abd-El-Barr, Muhammad M; Pennesi, Mark E; Saszik, Shannon M et al. (2009) Genetic dissection of rod and cone pathways in the dark-adapted mouse retina. J Neurophysiol 102:1945-55|
|Brill, Elliott; Malanson, Katherine M; Radu, Roxana A et al. (2007) A novel form of transducin-dependent retinal degeneration: accelerated retinal degeneration in the absence of rod transducin. Invest Ophthalmol Vis Sci 48:5445-53|
|Rosenzweig, Derek H; Nair, K Saidas; Wei, Junhua et al. (2007) Subunit dissociation and diffusion determine the subcellular localization of rod and cone transducins. J Neurosci 27:5484-94|
|Srinivasan, Vivek J; Ko, Tony H; Wojtkowski, Maciej et al. (2006) Noninvasive volumetric imaging and morphometry of the rodent retina with high-speed, ultrahigh-resolution optical coherence tomography. Invest Ophthalmol Vis Sci 47:5522-8|
Showing the most recent 10 out of 22 publications