Below are the specific aims I propose to learn more about ADOA type 3. Aim 1 focuses on a genomic approach, Aim 2 looks at chemical genetics and Aim 3 is a proteomic technique.
Autosomal Dominant Optic Atrophy (ADOA) type 3 is a rare disease that causes progressive, bilateral vision loss in children and infants. This clinically heterogeneous disease can cause decreased vision and cataracts as early as birth or in childhood and can progress with age. Additionally, neurological symptoms such as tremors, coordination problems and hearing loss are observed [1]. This form of ADOA is caused by OPA3, the gene encoding a mitochondrial membrane protein, named optic atrophy 3, that is involved in mitochondrial membrane dynamics (fission/fusion), apoptosis, and lipid metabolism [2],[3],[4]. The molecular function of OPA3 is not well understood and more research is needed to understand how dominant mutations in OPA3 can cause optic atrophy and why there is a large variation in disease severity.
The overall goal is to characterize the role of OPA3 in mitochondrial homeostasis in the retina. My hypothesis is that OPA3 plays an important role in mitochondrial fission, fusion and overall morphology in the retinal ganglion cells that contributes to proper energy production. I will be using Danio rerio as model organisms to elucidate the role OPA3 has in the mitochondrial membrane as these organisms are popular for eye related [5]. The long-term goal is to determine the mechanism by which OPA3 influences mitochondrial morphology and how gene interactions influence the severity of mitochondrial defects in ADOA type 3.
Aim 1 - Identify domains in OPA3 that are related to mitochondrial membrane dynamics
Hypothesis: I hypothesize that at least one domain in OPA3 contributes to mitochondrial morphology, specifically fission, fusion and fragmentation.
Rationale: OPA3 has low-complexity and determining the function of the domain could be useful in determining how mitochondrial membrane dynamics are altered.
Approach: I will use Pfam to predict potential functional domains in the human and zebrafish OPA3 gene. Using the discovered domains and the known ADOA type 3 mutations that cause optic atrophy [7], the CRISPR/Cas9 system will be used to introduce corresponding mutations into flies to observe changes in the mitochondrial morphology in mutants versus the wild type in retinal ganglion cells.
Aim 2 -Identify small molecules associated with the phenotypic rescue of OPA3 mutants
Hypothesis: There will be small molecules that inhibit mitochondrial fragmentation and optic atrophy by influencing mitochondrial membrane dynamics.
Rationale: The mechanistic role of OPA3 in mitochondrial fragmentation and membrane dynamics is unknown. Identifying small molecules that rescue optic atrophy and mitochondrial morphology, we can uncover insights into the mechanism of disease and potential treatments.
Approach: I will treat mutant zebrafish who carry mutations in the OPA3 gene causing ADOA, from aim 1, with a chemical genetic screen of small molecules to rescue the main phenotype of optic atrophy. The goal is to identify small molecules that influence optic atrophy and mitochondrial morphology in the retina. Optic atrophy will be measure by retinal ganglion cell death and overall health, while mitochondrial morphology will be assayed using mitochondrial staining and fusion/fission assays.
Aim 3- Identify proteins that interact with WT and mutant OPA3 proteins in the retina
Hypothesis: Mutant OPA3 protein that causes mitochondrial membrane defects will have novel or absent protein-protein interactions compared to WT OPA3 protein.
Rationale: There are no studies on OPA3 protein interactions within retinal ganglion cells with respect to mitochondrial membrane dynamics. By understanding the differences between the interactions between WT and mutant OPA3 proteins in the eye, we might better understand the mechanism by which mitochondrial membrane dynamics are influenced.
Approach: I will use TurboID to identify protein-protein interactions with OPA3 and its domains. Transgenic Zebrafish will be made to carry TurboID tagged OPA3, one group will have the WT OPA3 gene, while the others will carry a version of the mutant OPA3 causing ADOA, as described in aim 1. Proteins will be extracted from retinal tissues treated by biotinylating and antibody enrichment before being identified by liquid chromatography and tandem mass spectroscopy. The identified proteins will be categorized by GO terms and their related pathways.
The overall goal is to characterize the role of OPA3 in mitochondrial homeostasis in the retina. My hypothesis is that OPA3 plays an important role in mitochondrial fission, fusion and overall morphology in the retinal ganglion cells that contributes to proper energy production. I will be using Danio rerio as model organisms to elucidate the role OPA3 has in the mitochondrial membrane as these organisms are popular for eye related [5]. The long-term goal is to determine the mechanism by which OPA3 influences mitochondrial morphology and how gene interactions influence the severity of mitochondrial defects in ADOA type 3.
Aim 1 - Identify domains in OPA3 that are related to mitochondrial membrane dynamics
Hypothesis: I hypothesize that at least one domain in OPA3 contributes to mitochondrial morphology, specifically fission, fusion and fragmentation.
Rationale: OPA3 has low-complexity and determining the function of the domain could be useful in determining how mitochondrial membrane dynamics are altered.
Approach: I will use Pfam to predict potential functional domains in the human and zebrafish OPA3 gene. Using the discovered domains and the known ADOA type 3 mutations that cause optic atrophy [7], the CRISPR/Cas9 system will be used to introduce corresponding mutations into flies to observe changes in the mitochondrial morphology in mutants versus the wild type in retinal ganglion cells.
Aim 2 -Identify small molecules associated with the phenotypic rescue of OPA3 mutants
Hypothesis: There will be small molecules that inhibit mitochondrial fragmentation and optic atrophy by influencing mitochondrial membrane dynamics.
Rationale: The mechanistic role of OPA3 in mitochondrial fragmentation and membrane dynamics is unknown. Identifying small molecules that rescue optic atrophy and mitochondrial morphology, we can uncover insights into the mechanism of disease and potential treatments.
Approach: I will treat mutant zebrafish who carry mutations in the OPA3 gene causing ADOA, from aim 1, with a chemical genetic screen of small molecules to rescue the main phenotype of optic atrophy. The goal is to identify small molecules that influence optic atrophy and mitochondrial morphology in the retina. Optic atrophy will be measure by retinal ganglion cell death and overall health, while mitochondrial morphology will be assayed using mitochondrial staining and fusion/fission assays.
Aim 3- Identify proteins that interact with WT and mutant OPA3 proteins in the retina
Hypothesis: Mutant OPA3 protein that causes mitochondrial membrane defects will have novel or absent protein-protein interactions compared to WT OPA3 protein.
Rationale: There are no studies on OPA3 protein interactions within retinal ganglion cells with respect to mitochondrial membrane dynamics. By understanding the differences between the interactions between WT and mutant OPA3 proteins in the eye, we might better understand the mechanism by which mitochondrial membrane dynamics are influenced.
Approach: I will use TurboID to identify protein-protein interactions with OPA3 and its domains. Transgenic Zebrafish will be made to carry TurboID tagged OPA3, one group will have the WT OPA3 gene, while the others will carry a version of the mutant OPA3 causing ADOA, as described in aim 1. Proteins will be extracted from retinal tissues treated by biotinylating and antibody enrichment before being identified by liquid chromatography and tandem mass spectroscopy. The identified proteins will be categorized by GO terms and their related pathways.
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This web page was produced as an assignment for Genetics 564, a capstone course at UW-Madison.
References
[1] Bagli, E., Zikou, A., Agnantis N., et al. Μitochondrial Membrane Dynamics and Inherited Optic Neuropathies. (2017). In Vivo, 31(4), 511–525. https://doi.org/10.21873/invivo.11090
[2] Ryu, S.-W., Jeong, H. J., Choi, M., Karbowski, M., & Choi, C. (2010). Optic atrophy 3 as a protein of the mitochondrial outer membrane induces mitochondrial fragmentation. Cellular and Molecular Life Sciences, 67(16), 2839–2850. https://doi.org/10.1007/s00018-010-0365-z
[3] Wang, Y., Ying, X., Wang, Y., Zou, Z., Yuan, A., Xiao, Z., Geng, N., Qiao, Z., Li, W., Lu, X., & Pu, J. (2023). Hydrogen sulfide alleviates mitochondrial damage and ferroptosis by regulating OPA3–NFS1 axis in doxorubicin-induced cardiotoxicity. Cellular Signaling,107, 110655.https://doi.org/10.1016/j.cellsig.2023.110655
[4] Wells, T., Davies, J. R., Guschina, I. A., Ball, D. J., Davies, J. S., Davies, V. J., Evans, B. A. J., & Votruba, M. (2012). Opa3, a novel regulator of mitochondrial function, controls thermogenesis and abdominal fat mass in a mouse model for Costeff syndrome. Human Molecular Genetics, 21(22), 4836–4844. https://doi.org/10.1093/hmg/dds315
[5] Fadool, J., & Dowling, J. (2008). Zebrafish: A model system for the study of eye genetics. Progress in Retinal and Eye Research, 27(1), 89–110. https://doi.org/10.1016/j.preteyeres.2007.08.002
[6] Han, D., Dong, X., Zheng, D., & Nao, J. (2020). MiR-124 and the Underlying Therapeutic Promise of Neurodegenerative Disorders. Frontiers in Pharmacology, 10, 1555. https://doi.org/10.3389/fphar.2019.01555
[7] Grau, T., Burbulla, L. F., Engl, G., Delettre, C., Delprat, B., Oexle, K., Leo-Kottler, B., Roscioli, T., Krüger, R., Rapaport, D., Wissinger, B., & Schimpf-Linzenbold, S. (2013). A novel heterozygous OPA3 mutation located in the mitochondrial target sequence results in altered steady-state levels and fragmented mitochondrial network. Journal of Medical Genetics, 50(12), 848–858. https://doi.org/10.1136/jmedgenet-2013-101774