There remains an urgent need to prevent vision loss from diabetic retinopathy (DR), a common and significant problem in patients with diabetes. Preclinical studies have highlighted retinal oxidative stress in the pathogenesis of DR. We reason that identifying the retinal mechanisms mediating pathogenic oxidative stress in diabetes will be important in the development of new and effective treatments. In neurons, oxidative stress and L-type calcium channels (LTCCs) activity are closely linked: increased LTCC activity elevates intracellular calcium content which must then be removed against a steep inter/extracellular calcium concentration gradient via ATP-dependent mechanisms which generate oxygen free radicals. We have discovered that diabetes profoundly changes the overall activity of photoreceptor LTCCs. Remarkably, a non-anti-oxidant and photoreceptor-specific treatment that corrected the abnormal outer retinal LTCC phenotype in vivo concurrently eliminated diabetic retinal oxidative stress. These experiments, using a validated and analytical tool (MEMRI), provide the first evidence for an unexpected and potentially very important discovery linking abnormal photoreceptor LTCCs and oxidative stress in diabetes. In the retina there are 3 major LTCC subtypes (Cav): Cav1.2 (located in inner retina), Cav1.3 (in inner retina, photoreceptor, and retinal pigment epithelium layers), and Cav1.4 (only in photoreceptors. How diabetes alters photoreceptor subtype channels are not yet known. We hypothesized that diabetes induces reproducible abnormalities in photoreceptor Cav1.3 and 1.4 expression and activity, and these changes actively contribute to oxidative stress and diabetic retinopathy. To begin to evaluate our new working hypothesis, we propose the following specific aim, which will take full advantage of our expertise in blending high resolution and analytical in vivo imaging of photoreceptor pathophysiology (manganese-enhanced MRI) with biochemistry (reactive oxygen species, western blot analysis (channel expression)) and histopathology (trypsin digest) in models of DR. SA 1: Test that diabetic alterations in photoreceptor Cav1.3, and 1.4 LTCC activity are required for oxidative stress and DR. The proposed experiments will open up a new line of scientific inquiry that is expected to improve our understanding about the origins of oxidative stress in DR. Because Cav1.3 and 1.4 subtypes are relatively insensitive to commonly used calcium channel blockers, our specific genetic and pharmacological approaches in concert with a novel imaging technique make these studies highly significant. These experiments will be crucial in the identification of new molecular targets for prevention and treatment of DR. The proposed research is highly innovative because it investigates a previously unsuspected but key role of photoreceptor LTCC subtype abnormalities in diabetes-induced oxidative stress, and will thus establish a firm scientific basis for changing current thinking regarding the origins of, and therapeutic options for, pathogenic oxidative stress in DR.
The proposed research is relevant to public health because it will improve our understanding of the role of photoreceptor calcium channels in diabetic retinopathy and thus improve therapeutic options for preventing and controlling retinal morbidity. The proposed studies will introduce methodological and conceptual innovations that are expected to be highly relevant to NEI's mission of eliminating vision loss. Also, because calcium channels are at the cross-roads of how many cell types communicate and make decisions, future application of our new experimental approach in other complications of diabetes, and in other retinopathies, future application of our new experimental approach in other complications of diabetes, and in other retinopathies, are anticipated and will highlight new areas for improving therapeutic options..
|Berkowitz, Bruce A; Lewin, Alfred S; Biswal, Manas R et al. (2016) MRI of Retinal Free Radical Production With Laminar Resolution In Vivo. Invest Ophthalmol Vis Sci 57:577-85|
|Jo, Andrew O; Lakk, Monika; Frye, Amber M et al. (2016) Differential volume regulation and calcium signaling in two ciliary body cell types is subserved by TRPV4 channels. Proc Natl Acad Sci U S A 113:3885-90|
|Berkowitz, Bruce A; Schmidt, Tiffany; Podolsky, Robert H et al. (2016) Melanopsin Phototransduction Contributes to Light-Evoked Choroidal Expansion and Rod L-Type Calcium Channel Function In Vivo. Invest Ophthalmol Vis Sci 57:5314-5319|
|Berkowitz, Bruce A; Bredell, Bryce X; Davis, Christopher et al. (2015) Measuring In Vivo Free Radical Production by the Outer Retina. Invest Ophthalmol Vis Sci 56:7931-8|
|Berkowitz, Bruce A; Murphy, Geoffrey G; Craft, Cheryl Mae et al. (2015) Genetic dissection of horizontal cell inhibitory signaling in mice in complete darkness in vivo. Invest Ophthalmol Vis Sci 56:3132-9|
|Berkowitz, Bruce A; Gorgis, Jawan; Patel, Ankit et al. (2015) Development of an MRI biomarker sensitive to tetrameric visual arrestin 1 and its reduction via light-evoked translocation in vivo. FASEB J 29:554-64|
|Berkowitz, Bruce A; Grady, Edmund Michael; Khetarpal, Nikita et al. (2015) Oxidative stress and light-evoked responses of the posterior segment in a mouse model of diabetic retinopathy. Invest Ophthalmol Vis Sci 56:606-15|
|Kern, Timothy S; Berkowitz, Bruce A (2015) Photoreceptors in diabetic retinopathy. J Diabetes Investig 6:371-80|
|Saliba, Alexandra; Du, Yunpeng; Liu, Haitao et al. (2015) Photobiomodulation Mitigates Diabetes-Induced Retinopathy by Direct and Indirect Mechanisms: Evidence from Intervention Studies in Pigmented Mice. PLoS One 10:e0139003|