Current & Future Projects
In recent years, our group has made important breakthroughs by identifying two bisretinoid-mediated pathogenic pathways in the RPE, both having significant clinical and therapeutic impact for recessive Stargardt Disease (STGD1) and a sub-group of AMD patients. Firstly, we discovered that the ABCA4 gene responsible for STGD1 is expressed in the RPE cells in addition to the photoreceptor cells. Additional studies from our laboratory indicated that the bisretinoid-mediated complement dysregulation in the RPE cells plays a critical role in visual loss of STGD1 and late-onset macular degenerations.
Our future research involves innovative and translational studies directed towards dissecting the intercellular organelle dynamics in the RPE cells with a focus on lysosomes maturation, recycling of retinoids and lipids relevant to cell bioenergetics, identifying complement membrane-mediated signaling pathways responsible for the loss of RPE, and investigating the role of epigenetic factors to aging under normal and pathological status.
i. Complement dysregulation in the RPE cells is a key pathogenic pathway in Stargardt disease (STGD) and Age-related Macular Degeneration (AMD)
Our developed gene-therapy approach established a ‘proof of principle’ that sub-lethal attack on the RPE by the complement leads to loss of the photoreceptors in the Abca4-/- mouse model and presumably in STGD1 patients. Furthermore, our data indicate that dysregulation of the complement system is a common etiological pathway for STGD1 and AMD, the leading cause of visual loss in the elderly. We initiated two orthogonal approaches to investigate the role of the complement system in human ocular tissue using: (1) biochemical and morphological characterization of post-mortem donor eye tissue samples from patients clinically diagnosed as STGD (Figure 1; Hu et al, Redox Biology 2020); and (2) developed a novel “disease-in-a-dish” system using intrinsically pluripotent stem cells (iPSC) obtained from the skin biopsy of the clinically and molecularly diagnosed STGD1 patients and control individuals without ABCA4 variants.
Figure 1
Hu et al, Redox Biology 2020
Retinal Pigment Epithelium in Stargardt Disease (STGD): evaluation of human eye donors.
Abbreviations: BrM = Bruch’s Membrane; C3b = C3 complement fragment; CC = Choroid capillaries; L = Lipofuscin; ML = Melanolipofuscin; MV = Microvilli; N = Nucleus; TJ = Tight junctions
ii. ABCA4 is expressed in the RPE cells in addition to the photoreceptors: implications for STGD1 patients
We discovered that ABCA4 is additionally present in the RPE cells where it co-localizes with endolysosomal proteins. Demonstration that ABCA4 is expressed in the RPE, and that loss of ABCA4 from these cells plays an important role in recycling of retinoids, has vital implications for the treatment of STGD1 patients. Our findings suggest that RPE cells, in addition to the photoreceptors, should also be targeted in rescue approaches to treat all ABCA4-mediated retinal degenerations. These data were published in the Proceedings of National Academy of Sciences (Figure 2; Lenis et al, PNAS 2018). In the light of this discovery, we want to investigate the contribution of each cellular component in developing of STGD1 pathogenesis. To address this issue, we generated ABCA4 transgenic and conditional knockout mice to assess the function of ABCA4 independently in the RPE and photoreceptor cells.
Figure 2
Lenis et al, PNAS 2018
0ABCA4 mRNA is expressed in human and mouse retinal pigment epithelium cells in addition to the photoreceptors. In situ hybridization using the RNAscope chromogenic assay with a species specific ABCA4 probe on cadaveric human retina section (left) and mouse retina of wild-type (Abca4+/+, middle) or knockout (Abca4-/-, right) mice. Note the intense chromogenic reactivity (red punctate staining, indicated by the black arrows) for ABCA4 mRNA in retinal pigment epithelium cells and outer nuclear layer of the photoreceptor cells. This reactivity is absent in the retina of Abca4-/- mouse.
Abbreviations: CC = Choroid capillaries; RPE = retinal pigment epithelium; OS = outer segments; IS = inner segments; ONL = outer nuclear layer; INL = inner nuclear layer
iii. Development of a new models to study Age-related Macular Degeneration (AMD)
Other studies in my lab are focused on ageing processes in the RPE using complex genetic mouse models with an emphasize on the complement system and lipid/retinoid metabolism. Towards this goal, we generated mouse lines lacking two or three genes associated with macular degenerations (ABCA4, CFH, ELOVL4, etc). These new mouse models are additional tools to interrogate the synergistic pathways of formation of bisretinoids, complement dysregulation, lipid metabolism and mitochondria bioenergetics in the RPE cells. These mouse studies are complemented by experiments using human primary cultured iPSC-derived RPE cell lines with known disease-associated mutations.
iv. Epigenetics in Stargardt disease and Age-related Macular Degeneration (AMD)
Despite recent links of epigenetics to aging and disease status, a specific epigenetic-mediated mechanism responsible for the RPE cellular death has not yet been identified. Our preliminary data supports a role of the specific epigenetic factor to mediate mitochondria dynamics in aging and STGD1 RPE cells. Rescue studies and new models to validate this pathway are currently developing in the lab.
