Department of Ophthalmology
West Virginia University School of Medicine
Morgantown, WV

BASIC RESEARCH PROJECT

Target NAD degradation in age-related macular degeneration

Research Interests

Scientific Summary: Age-related macular degeneration (AMD) is the leading cause of blindness among the elderly. There are currently no effective treatments available for “dry” or atrophic AMD, which includes the earlier loss of retinal pigment epithelial (RPE) cells. Dr. Du recently used an unbiased approach to study the nutritional consumption in human RPE cells and found that RPE prefers to use an amino acid, proline, to support its energy metabolism. Interestingly, RPE converts the proline into key intermediates in metabolism and exports these intermediates to fuel the retina. The preliminary data confirm that the RPE supports retinal energy metabolism through proline in vivo and supplementation of proline in RPE or diet could protect oxidative damage and improve age-related visual decline. Consistently, the mutations of genes associated with proline metabolism in patients have been known to cause retinal degeneration. Findings and published results suggest that proline metabolism is fundamentally important to the pathogenesis of AMD.

The objective is to study how this special amino acid is associated with retinal function and degeneration, and to test the treatment with proline on an AMD-like disease model. Study findings will allow Dr. Du to better understand how energy metabolism impacts retinal degeneration and provide the foundation for developing novel nutrition-based therapies for AMD.

Plans for 2024

The purpose of Dr. Du’s project is to study how NAD+, a critical molecule controlling metabolism, breaks down in both healthy and diseased retina and retinal pigment epithelium (RPE). Dr. Du wants to understand if stopping this breakdown can protect retinal degeneration.

In the next period, The Du laboratory will focus on understanding how NAD+ breaks
down in the RPE of older mice by quantifying NAD+ degradation pathway in RPE from
the aged mice in vitro and in vivo. The team will compare how key enzymes involved in this breakdown process are expressed in younger and older mice. Additionally, they will continue to assess how NAD+ degradation pathways contribute to oxidative damage, RPE survival, visual function and retinal degeneration.

Specific Aims:
Aim 1: Determine the change of NAD+ breakdown in old RPE. We will study whether aging increases NAD+ breakdown in RPE to decrease the levels of NAD+ and its related molecules, which are critical to maintain metabolism. They will measure how quickly NAD+ is degraded and examine levels of specific enzymes NAD+ degradation in young and old mice.

Aim 2: Evaluate the role of NAD+ breakdown in the protection of RPE-initiated retinal degeneration. They will assess whether blocking NAD+ breakdown will raise the levels of NAD+ and its related metabolites to protect against RPE death, visual decline and retinal degeneration by oxidative damage in an AMD-like mouse model.

Progress in 2023

In the first year of Dr. Du’s new pilot project, significant progress was made. Dr. Du found NAD+  primarily breaks down in the RPE layer in both mice and human. NAD+ breaks down differently in male and female eyes. Further, his team also developed methods to quantify the metabolic flux of NAD+ breakdown in vivo in mice. By deleting CD38, a key enzyme in NAD+ breakdown, we were able to increase the levels of NAD+, NADH, NADPH, glutathione and ATP in the RPE. Furthermore, deleting CD38 protects oxidative damage and glial activation in the in the AMD-like mouse model suggests that targeting NAD+ degradation could be a promising approach. They presented some of these findings at the ISER 2023 conference and published two papers related to this project in scientific journals (IOVS and JBC).

2023 Publications

The research goal in 2023 is to investigate the role of NAD+ degradation in the healthy and diseased retina, and evaluate the protection of retinal degeneration by inhibition of NAD+ degradation pathways.

Specific Aims:
Aim 1: To measure the rates of NAD+ degradation and synthesis in young and aged RPE and retina by testing the hypothesis that RPE is the primary site of NAD+ degradation that is enhanced in aging. The lab will measure the concentrations of metabolites in NAD+ metabolism, and measure the rates of NAD degradation and synthesis using stable isotope tracers coupled with mass spectrometry in young and aged mice.

Aim 2: To evaluate the treatment of acute AMD-like mouse models by inhibiting NAD+ degradation. We will test the hypothesis that inhibition of NAD+ degradation will boost NAD+ metabolism to protect against oxidative damage and retinal degeneration. Dr. Du will use genetic and pharmacologic approaches to inhibit NAD+ degradation and evaluate their protective effects on retinal function and degeneration in AMD-like mouse models.

Progress in 2022

Nutritional strategies in Age-related Macular Degeneration

The purpose of this portion of the project was to study the role of proline metabolism in retinal function and viability, and to develop nutritional approach to treat AMD.

Dr. Du made significant progress in 2022, finding that proline dehydrogenase (PRODH) is critical for the metabolic communication between the retinal pigment epithelium (RPE) and the neural retina. PRODH deficiency blocks the export of proline-derived nutrients from RPE to the retina. PRODH is critical for both carbon metabolism in the mitochondrial Krebs cycle and nitrogen metabolism in producing neurotransmitters and amino acids in the retina. PRODH knockout mice have age dependent visual decline. Dr. Du had two manuscripts published in 2022.

Specific Aims: Aim 1: Determine the role of proline catabolism in retinal metabolism and degeneration. We will test the hypothesis that RPE-enriched PRODH is responsible for the support of retinal metabolism by proline and the deletion of PRODH will make the retina vulnerable to retinal degeneration. We will measure retinal metabolism, visual function and retinal degeneration in an acute AMD-like model in PRODH knockout mice with regular or proline-rich diets. Aim 2: Determine the protection of retinal degeneration by enhancing proline catabolism. We will test the hypothesis that enhancing proline catabolism supplies the retina with more intermediates for mitochondrial metabolism and anti-oxidants to protect against retinal degeneration. We will test the protection of visual function and retinal viability in an acute AMD-like model by subretinal injection of AAV PRODH.

Progress in 2021

Dr. Du’s laboratory made significant progress on both Aims of his project. He found RPE specific proline transporter, SLC6A20A is important for retinal energy metabolism and visual function. RPE could utilize the dietary proline to produce multiple crucial amino acids to nourish the neural retina including glutamate, aspartate and serine. Mice fed with proline-free diets for 8 months show decreased visual function, suggesting that proline is important to maintain retinal metabolism and function.

Some of Dr. Du’s findings were presented at ARVO 2021 and published in Bio Protocol. Dr. Du’s team continued the diet supplements for two more months to test the protection of proline on age-related visual decline and metabolic dysfunction and prepare manuscripts for publication.

Specific Aims: Aim 1: Determine the role of proline transport in retinal metabolism and health in vivo.  The research team will test the hypothesis that the proline transporter SLC6A20 is responsible for proline utilization to fuel retinal metabolism and maintain retinal health. Dr Du’s team will measure metabolic, functional, and morphological changes in the inducible RPE-specific knockout of SLC6A20 in mice. Aim 2: Determine the role of dietary proline in retinal metabolism and its protection of age-related visual decline.  Dr. Du’s research will test the hypothesis that dietary proline will boost retinal mitochondrial metabolism to protect against age-related retinal degeneration.  They will determine the role of proline-enriched and proline-deficient diets on retinal mitochondrial metabolism, visual function, and retinal morphology in mice.

Progress in 2020

Dr. Du made significant progress in 2020.  His research team generated inducible RPE-specific SLC6A20A knockout mice and whole body SLC6A20A mice and found that SLC6A20 and other proline metabolism genes are highly upregulated in the RPE.  The inhibition of mitochondrial respiration in RPE blocked proline utilization to disrupt the nutrient transport to the retina.   Additionally, Dr. Du’s research found a proline-enriched diet increases the biosynthesis of glutothione and NADPH, protects oxidative damage, and improves visual function in an acute AMD-like mouse model. Dr. Du’s research resulted in six publications in 2020.

Progress in 2019

In 2019, Dr. Du found proline protects retinal pigment epithelium (RPE) against oxidative damage both in vitro and in vivo. Dietary proline supplement improves visual function in an AMD-like mouse model.  These new findings were presented at ARVO 2019 in Vancouver and were also published.  Additionally, Dr. Du generated floxed mice targeting proline transporter SLC6A20 and validated a new inducible RPE CRE mouse line.

Results Previously Published: Dr. Du’s research team and other groups have published results showing that proline metabolism is linked to retinal degeneration, SLC6A20 expresses almost exclusively in RPE with its expression correlated with proline consumption, and proline is an important nutrient in energy metabolism, anti-aging and stress protection.