Department of Cellular Biology & Anatomy

Medical College of Georgia, Augusta University

Augusta, GA

BASIC RESEARCH PROJECT

The roles of RIP kinase 3 in the development of AMD-like pathologies during cytomeglavirus ocular latency

Research Interests

Precise events contributing to Age-related Macular Degeneration (AMD) development remain uncertain; yet several immunological/inflammatory mechanisms are implicated. Autophagy and phagocytic function are highly correlated with AMD. RIP kinases play various tissue-specific roles in cell death, innate immunity and autophagy. Human cytomegalovirus (HCMV), which infects 50 to 80% of individuals in the human population, may be a risk factor for AMD progression. The eye is a major target organ for congenital/newborn HCMV infection and the choroid/RPE may be a site of HCMV latency. Our central hypothesis is that latent HCMV infection in the choroid/RPE contributes to the pathogenesis of AMD, thereby presenting a novel target for treatment.

Plans in 2023

The purpose of this project is to test the hypotheses that RIP3 contributes to the death/degeneration of ocular cells/tissues and development of AMD–like pathologies via the production of inflammatory factors, activation of cell death pathways, and via cross-talk among cell death, autophagy and LC3-associated phagocytosis  (LAP) during MCMV ocular latency.

In 2023, we will continue to use a novel mouse model of systemic neonatal (MCMV) infection as well as several strains of conditionally RIP3 depleted mice in which RIP3 is depleted in either capillary endothelia, RPE, or macrophage/microglia to investigate the role of RIP3 in the development of Age-related macular degeneration (AMD)-like pathologies during MCMV ocular latency. Secondly, using isolated retinal microglia from RIP3^-/- mice and wild type RIP3^+/+ mice to test the hypothesis that RIP3 plays dual roles in innate immunity of microglia: pro-inflammation via activation of activation of inflammasomes/NFkB and anti-inflammation via decreased expression of IL1 receptor.

Progress in 2022

Our studies show that: (1) latent ocular MCMV infection following systemic neonatal MCMV infection is associate with development of retinal and choroidal pathologies with some features of human AMD, while  inflammatory factors/pathways including RIP3, IL1, NFkB were activated and multiple cell death pathways, including caspase-3 dependent apoptosis, necrosis and RIP3 associated necroptosis are activated and contribute to degeneration of photoreceptors, RPE and choroidal capillaries; a draft of manuscript based on these results has been submitted to IOVS for review;  (2) although MCMV DNA and mRNA transcripts of several MCMV latency-related genes were detected in eyes and extraocular tissues of both RIP3^+/+ and RIP3^-/- mice 12 months following systemic neonatal MCMV,  the mean retinal thickness was significantly lower in eyes of latently infected RIP3^-/- mice, compared to eyes of latently infected RIP3^+/+ mice. In addition, two strains of RIP3 conditional ko mice in C57BL/6 background including tamoxifen-inducible, RPE-specific, RIP3 depleted mice (RIP3^flox/flox; Tyrosinase-Cre/ERT2+) and tamoxifen-inducible, macrophage/microglia-specific, RIP3 depleted mice (RIP3^flox/flox; CX3CR1/ERT2+) were developed and we are in the progress to cross these mice into BALB/c background for 6 generations and used for proposed studies.

Dr. Zhang’s Previous RRF Research Project, 2013-2015

Previous Research Interests

Herpes simplex virus (HSV) is believed to be the leading cause of infectious blindness in the developed world. Many aspects about ocular herpesvirus infections, in general, and about acute retinal necrosis (ARN), in particular, in human patients remain enigmatic. HSV-1 inhibits autophagy through Beclin-binding domain (BBD) of ICP34.5. The aims of this project are 1) to determine if inhibition of autophagy by HSV-1 plays a key role in ocular HSV-1 infection by inhibiting innate immunity mediated by NLRP3 inflammasome and 2) to determine if inducer of autophagy or the NLRP3 inflammasome is beneficial to the control of HSV-1 retinal infection.

Progress in 2015

Dr. Zhang’s lab attempted to extend upon findings to investigate the relationship between autophagy and the NLRP3 inflammasome. By using inflammasome deficient mice, they will investigate if the inflammasome participates in early innate immune response against ocular HSV-1 infection and determine if depletion of the NLRP3 inflammasome can reverse the inhibition of virus spread and replication and enhanced innate immune responses observed in BBD deficient, HSV-1 infected eyes. In addition, his group will continue their studies on Tat-beclin 1 to investigate if this autophagy inducing peptide can override the inhibition of autophagy and subsequent innate immune responses mediated by the BBD of wild type HSV-1.

Progress in 2014

Dr. Zhang’s team has published three papers in top international journals in 2014. These publications report new findings on mechanism of cell death of infected and uninfected retinal neurons as well as new methods to control retinal viral infection. By using mice deficient for autophagy specifically in neural cells, his group demonstrated that HSV-1 inhibits autophagy and subsequent activation of the NLRP3 inflammasome via Beclin-binding domain (BBD). Inhibition of autophagy by HSV-1 BBD increases death of infected neurons by apoptosis. Their results also suggest that early administration of autophagy inducing peptide Tat-beclin 1 inhibits viral replication and protects retinal cell death by apoptosis.

Progress in 2013

The primary objective of Dr. Zhang’s research program is to understand the pathogenesis of herpes simplex virus 1 induced acute retinal necrosis (ARN) and cytomegalovirus retinitis by using mouse models and organatypic retinal culture models. Currently his group is studying the mechanism by which autophagy activates the innate immune response during HSV-1 retinal infection and how autophagy balance the beneficial and harmful effects of inflammatory host responses by interacting with NLRP3 inflammasome components. His lab is exploring methods to control ocular HSV-1infection by stimulating anti-HSV-1 innate immune responses and by reducing neuronal cell death by apoptosis. He published his results in Journal of Neuroimmunology in 2013.