Throughout the year, Department of Ophthalmology faculty members welcome medical student involvement in research projects. Medical students interested in pursuing research in the Department of Ophthalmology should review the topics below and contact the faculty member directly.
Optimization of an IkBa destabilized domain to achieve enhanced degradation kenetics and in vivo regulation of inflammation
Mentor: John D. Hulleman, Ph.D.
We have previously shown that appending a destabilized DHFR domain onto the natural inhibitor of NFkB, IkBa, can control its abundance and enable us to conditionally inhibit NFkB signaling in a small molecule-dependent manner through promoting DHFR-IkBa stabilization. However, under treatment conditions without the small molecule stabilizer of DHFR, trimethoprim (TMP), a small amount of DHFR-IkBa protein was observable in cultured retinal pigment epithelium (RPE) cells (which is why we imparted additional control over our strategy using a doxycycline-inducible plasmid). The small amount of DHFR-IkBa that exists in the absence of TMP can still minimally inhibit NFkB signaling, which is sub-optimal. In order to transition this strategy for use in in vivo models of retinal degeneration, and use a single small molecule (TMP) to accurately control NFkB signaling, we need to generate a version of DHFR-IkBa that can be degraded more quickly and efficiently by the ubiquitin proteasome system such that under conditions without TMP, our introduced version of destabilized domain IkBa is undetectable, yet strongly produced upon addition of TMP. Generation of such a regulatable version of DHFR-IkBa will allow us to effectively control inflammatory signaling in vivo.
The student who choses this project will use state-of-the-art cloning and protein engineering strategies to generate fully functional destabilized domain versions of IkBa that can be more effectively degraded by the ubiquitin proteasome system. We will alter the location of the DHFR destabilizing domain (N- vs. C-terminal) and the number of destabilized domain attached to IkBa. This student will then test the anti-inflammatory functionality of the newly developed destabilized domain-IkBa variants by using quantitative PCR (qPCR), western blotting and ELISAs. Ultimately, after validation of an optimized destabilized version of IkBa, the student will then generate a DHFR-IkBa AAV vector for in vivo use. This AAV vector will be subsequently packaged and used to prevent inflammation-associated retinal degeneration in a Stargardt retinal dystrophy model (in later studies).
Development and validation of a conditional approach to promote lysosomal biogenesis and lipid/protein catabolism in retinal cells
Mentor: John D. Hulleman, Ph.D.
A number of inherited (e.g., Stargardt disease) and age-related eye diseases (e.g., age-related macular degeneration, AMD) are characterized by the accumulation of protein and lipid deposits inside the lysosome of retinal pigment epithelium (RPE) cells. While these deposits (called lipofuscin) can be considered a ‘normal’ hallmark of aging, if they are present in sufficient quantity or size in the retina, they serve as risk factors for the development of retinal degeneration and potentially blindness. Thus, identifying ways of promoting proper lipid catabolism in the RPE may serve as an effective strategy to thwart lipid dysregulation and the onset of pathogenic lipofuscin, and possibly another hallmark of AMD, protein and lipid-rich sub-RPE deposits called drusen.
The lysosomal-autophagic pathway plays a critical role in lipid catabolism and cellular clearance. Recent studies have found that transcription factor EB (TFEB) is a gene that controls lysosomal biogenesis, autophagy and that it promotes transcriptional regulation of lipid catabolism genes. Thus, activating a transcription factor such as TFEB in RPE cells may serve as a unique and powerful approach to prevent lipid/protein dysregulation in diseases such as AMD or Stargardt disease. However, as with all signaling pathways within the cell, too much activation of pathways such those regulated by TFEB (i.e., autophagy) are counterproductive and likely detrimental. Therefore, we propose to generate a small molecule-regulated, genetically encoded version of TFEB (DHFR-TFEB) that allows us to turn its activity ‘on’ and ‘off’ at whim.
The student who undertakes this project will use cloning, site-directed mutagenesis and advanced cell biology techniques to identify a ‘super’ active version of TFEB. This student will then test whether we can subsequently regulate TFEB protein abundance by fusing it to a DHFR-based destabilized domain. Identification of a regulatable, ‘super’ active version of TFEB will serve as a lead candidate for the treatment of Stargardt disease using this unique approach.
Direct detection and quantification of the AREDS formulation in the mouse eye
Mentor: John D. Hulleman, Ph.D.
Dry age-related macular degeneration (AMD) is a complex and prevalent disease with very few potential treatments. Currently, the only suggestion by the Age Related Eye Disease Study (AREDS) for decreasing the chances of developing advanced atrophic AMD is to take daily doses of high levels of antioxidants (vitamins C and E), zinc, lutein and zeaxanthin. Presumably, these components are able to protect the retina through either indirect means (i.e., by altering the signaling throughout the body, which ultimately ‘trickles-down’ to the eye), or through direct effects on retina (i.e., are physically transported to the retina). Yet, it is unclear which of these scenarios is true. Furthermore, whether this supplementation can protect the retina in mouse experimental models of retinal degeneration is unknown.
Thus, we propose to administer the AREDS2 formulation to mice by oral gavage followed by quantification of the AREDS components in the mouse eye by mass spectrometry. Following these experiments, we will then assess whether the AREDS2 has any effect on basal antioxidant signaling in the mouse retina. Subsequently, we will then gauge whether pretreatment of mice with the AREDS formulation protects against light-induced retinal thinning and reduction in electroretinogram (ERG) signal.
The student who undertakes this project will become proficient in mouse handling, tissue processing for biochemical, molecular biology and histological analysis, and assessment of retinal function. Ultimately, completion of this project will advance our understanding of how the AREDS formula may affect the retina, and whether this supplementation can also protect in mouse models of retinal degeneration.
These projects will be posted midway through the 2017-18 academic year.