My Internship
Figure 1 shows an overview for the research being conducted at the Meyer Lab.
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Overview
I work with Dr. Jason Meyer at the Stark Neurosciences Research Institute (IUSM). My work revolves around Glaucoma research, and specifically the E50K mutation in the Optineurin gene. This mutation leads to a severe type of normal tension glaucoma in human patients.
Millions of people around the world live with neurodegenerative diseases, but interestingly, the mechanism for neurodegeneration is largely conserved throughout various forms of CNS diseases. Therefore, investigating glaucoma can help provide insights not just for glaucoma but also for other CNS diseases such as Parkinson's and Alzheimer's.
To further understand glaucomatous neurodegeneration, human pluripotent stem cells (hPSCs) are differentiated into neurons, and these neurons are used as models of disease. These mutated cells were found to display shorter neurites and decreased complexity when comparing the number of primary neurites, their length, and soma size.
Additionally, it is known that the OPTN gene is highly associated with mitochondria. Therefore, it is possible that the E50K mutation can disrupt mitochondria and cause insufficient power production in RGCs, which can cause many detrimental downstream effects.
The research
Abstract
Characterization of Glaucomatous Neurodegeneration of Human Pluripotent Stem Cell Derived Retinal Ganglion Cells With an Optineurin E50K Mutation.
Glaucoma is the leading cause of blindness with progressive degeneration in retinal ganglion cells (RGCs). Similar to neurons in central nervous system, RGCs are post mitotic and unlikely to regenerate after injury or degeneration. Our goal is to study the mechanism behind glaucomatous neurodegeneration and explore possibilities to protect RGCs from dying. To further understand glaucomatous neurodegeneration, human pluripotent stem cell (hPSCs)-derived RGCs (hPSC-RGCs) serve as an appropriate system to generate human RGCs and allow modeling of neurodegeneration. In this study, our initial efforts used a genome editing CRISPR/Cas9 approach to insert the Optineurin E50K mutation, a mutation leading to a severe type of normal tension glaucoma, in wild-type hPSCs. This was followed by differentiation to RGCs , and these cells were explored to quantify their neurite outgrowth and mitochondrial complexity. When comparing mutated cells with their isogenic control hPSC-RGCs, E50K mutation hPSC-RGCs displayed neurite shortening and decreased the complexity when quantifying the number of primary neurites, the length of neurites, and soma size. Future studies will identify targets that can protect RGC neurite outgrowth as a way of RGC neuroprotection in glaucomatous neurodegeneration.
To perform studies on retinal ganglion cells (RGCs), they must first be differentiated from human pluripotent stem cells (hPSCs). After differentiation, the RGCs are screened via immunostaining to ensure they have properly differentiated. Results for sample stains are shown below.
Figure 2 shows a retinal organoid at Day 80.
BRN3 - RGC marker | RCVRN - Retina protein marker | DAPI - Nucleus/DNA marker
Figure 3 shows a retinal organoid at Day 80. '
This sample is stained with BRN3:tdTomato.
Figure 4 shows one RGC section exposed to various types of fluorescence.
RBPMS - RGC marker | MAP2 - Dendrite marker | PAX6 - protein marker | ISL1 - protein marker
Characterization of a glaucomatous neurodegeneration model from hPSC-RGCs
Compared to the WT cells, E50K cells were found to have fever shorter neurites, less primary neurites, and possess a smaller soma. The image on the right shows a representative depiction of what was obtained.
Figure 5 shows a schematic for the difference in neurite growth between WT and OPTN (E50K) RGCs.
Figure 5 shows the schematic for the DNA mutation that occurs in the E50K mutation, in addition to the DNA sequencing results for various samples.
In the E50K mutation, there is a point mutation at the 50th amino acid which changes glutamic acid [E] to lysine [K]. The mutated amino acid is shown in red in figure 5.
Two additional silent mutations are added (G > C and G > A), also shown in red, to assist with PCR screening and to prevent Cas9 from cutting the donor template by modifying the PAM site.
Chromatography DNA sequencing is used to quantify the change in the nucleotide of interest.
Figure 6 shows the inverted fluorescent imaging for RGCs throughout various stages of development. Isogenic controls are shown from A-D, and the OPTN (E50K) cells are shown in E-H.
Figure 7 shows a schematic for neurite outgrowth from a soma.
Inverted fluorescent images (figure 6, A-H) help to view the neurites throughout various stages of the cells' development. A and E show that there is similar neurite complexity. However, significant neurite retraction in OPTN (E50K) RGCs became apparent at 4 weeks.
This is shown in a more intuitive way by figure 7. The neurites are drawn out using image analysis software, and it becomes more clear that the complexity in OPTN (E50K) RGCs is diminished significantly compared to the isogenic control.
Figure 8 shows four graphs with data regarding neurite shortening in Week 4 E50K RGCs (from Figure 7).
Figure 8 shows the quantification of data for Week 4 E50K RGCs compared to the isogenic control. While it is clear visually that there is a difference in complexity, these graphs provide a mathematical approach towards determining the significance of neurite shortening in E50K RGCs. Compared to the control, OPTN (E50K) RGCs are found to contain less neurites, less branch points, shorter neurites, and smaller soma size.
Less complexity in neurites has been associated with other CNS diseases besides glaucoma such as Parkinson's, ALS, and AD, all of which ultimately result from degeneration of neurons.
Learning and Skills
I already have experience in a laboratory setting, but never one in an academic institution. My experience was in industry before, and there is not as much liberty with projects as there is in academia. Given that my prior experience was at Lilly, there were also many legal obstacles regarding research. For instance, I was not able to present my research outside of Lilly due to legal concerns over their proprietary drug. As such, my presentations were internal with fellow Lilly scientists. In contrast, I have found that at the IUSM labs, people freely speak of their research and often give talks regarding what they are working on. These talks are often in the form of zoom meetings where the researchers present a PowerPoint over what they are doing. As such, there seems to be a lot more freedom to speak about what people are doing in academia compared to industry.
There are various techniques which I have performed in the Meyer lab that I had prior experience with. For instance, I performed PCR and gel electrophoresis, as well as general lab techniques such as aliquoting, microscopy, and pipetting. Many of the newer concepts and procedures performed in lab are things that I have gotten the opportunity to experiment with in classes such as Genetics Lab and Cell Biology Lab. These include Western blotting, enzyme digestion, cell quantification, cellular staining, and and immunoblotting . I have also learned many brand new things such as differentiation of pluripotent stem cells, fluorescent microscopy, DNA and protein concentration quantification, use of software such as ImageJ, Fiji, and Prism, and general maintenance of astrocytes. Most of what I have learned is regarding the preparation and growth of cell cultures because there is a lot that goes into creating and proliferating viable cell cultures of neurons. Overall, it is great to see that the things we are learning in class have practical applications in professional research laboratories.
Like mentioned prior, cultivation and maintenance of retinal ganglion cells from stem cells is a delicate and long process. Part of my work at the site involved maintenance of astrocytes. I would feed them retinal differentiation medium, neuronal medium, chop the astrocyte spheres that developed, change mediums as needed, and ensure that their morphology was appropriate for their stage of development. Taking care of the time intensive tasks allowed for the PhD student I was working with to focus more on other laborious tasks.
The Workplace
In terms of the atmosphere of the Meyer lab, I found that it is very pleasant and productive. I have found that the people I work with demonstrate a positive attitude, are kind, and work hard but also have a relaxed atmosphere. In general, as a professional, I want to keep working on my interpersonal relations. Having the ability to communicate with others in an efficient way is hugely important not just for medicine but for being a professional. Interpersonal relations is one of those things that everyone keeps working on throughout their whole life, but I would like to be in a good spot by the time I have my medical school interviews. In terms of the actual people in lab, I am not sure what I thought it would be like. I expected for me to be the only undergraduate in lab but it turns out that there are a few others, and I frequently interact with them, and have since become good friends with them. Additionally, there are all types of people present ranging from PhD post-docs to research technicians. As such, the environment is quite pleasant and varied. The lab itself is quite large, and we have two separate research rooms.
In terms of the day-to-day life, I was surprised by the amount of time and effort it takes to maintain cell cultures alive and proliferating. Often, people will have to come in on the weekends to feed their cells and keep them happy. Additionally, any amount of contamination can destroy a culture that has been growing for months. Therefore, a high degree of sterile technique is required in order to do anything with the cell cultures. Finally, it was surprising to me that the students took so much time out of their day working on various tasks other than experiments such as preparing slides for presentations and writing reports.
Overall, I wasn't quite sure of what to expect from this experience, but like I mentioned prior, I thought I would be the only undergraduate. The staff of the lab is very diverse and this is something that I enjoy thoroughly as everyone has a distinct story to tell regarding how they ended up at the Meyer lab. In terms of the work itself, I thought I would be doing monotonous activities but I have been pleasantly surprised. Working with the cells is a lot of fun, and though I have often achieved bad results, I still obtain good results and that sensation is great.
The lab is heavily focused on perpetual learning and many of the people I engage with are fellow students. As such, I can relate to them on multiple levels. My ideal workplace culture would be one where everyone understands their role and helps each other out. At my internship site, everyone is extremely knowledgeable on their research and readily answer any and all of my questions regarding what they are doing.
My attitudes and beliefs have changed dramatically due to the diversity of people in the lab. I love working with students who are older than me because I can see what a post-undergraduate life could look like. Additionally, I have been able to explore various cultures and enjoy new restaurants around Indy that I otherwise would likely not have known about. Everyone in the lab has various perspectives on life, but everyone is very nice and receptive to questions. I can say that I have grown as a professional because I appreciate people more now.
Successes and Challenges
Some of my favorite moments from lab are when cells undergo immunostaining, and then you are able to view specific sections of the retinal cells under various types of light. For example if a cell is stained with GFP, then the cells' mitochondria will fluoresce. Also, sometimes I am able to make gels and run them in gel electrophoresis. This is a success because running the gel and seeing the results is essentially the finale to see if all of the previous steps (DNA extraction/purification, PCR, etc.) all worked properly.
By far, the biggest challenge was learning the research that people are involved with. I am still actively learning the nuances as to what exactly people do and why they do certain things. I have learned a great deal about neuroscience and academic settings in general, partially because I do ask a lot of questions.
I will say, one of the best feelings is obtaining good results from a gel. The process of DNA extraction, quantification, PCR, purification, enzyme digestion, and ultimately running a gel takes many hours and several days. Likewise, it is very frustrating to see a gel without any bands. However, everything is a learning experience and if nothing else, I at least got more practice in the techniques used to prepare the samples. However, I have been successful at many things such as successfully feeding cells and making sure that they are alive and proliferating. Also, I have gotten good gel results in the past, and it is always a treat to be able to see cells under the microscope after I perform the immunostaining.
The biggest challenge is still understanding the research that is going on. I have since developed a much deeper understanding of what is happening, and it does not seem as convoluted as it once did. I ask many questions, and I would argue this aspect has helped me tremendously in my lab knowledge. Everyone in the lab focuses on a different area of neurodegenerative research, and as such, they all have various techniques that they use for their respective cells. For example, growing Astrocytes is different than growing Neurons, and as such requires distinct procedures to be utilized. One of the most useful things towards discovering the processes that are going on in lab has been reading the academic papers that have been published by members of the Meyer lab. By reading the papers, I have been able to understand the background of what we are doing and I was also able to come to lab with more questions which were readily answered.
