Sambuddha Basu, PhD, University of Central Florida, Burnett School of Biomedical Sciences, talks about research being done using a gene-editing technique called CRISPR to tag and study the gene strongly correlated to Parkinson’s disease.
Interview conducted by Ivanhoe Broadcast News in November 2017.
Explain what are you studying and how you came about it.
Basu: We work in a Parkinson’s disease lab. In this lab, all the aspects of Parkinson’s disease are actually being researched on. This paper is also about developing a particular tool in order to facilitate Parkinson’s research in terms of drug screening processes. What we essentially did is that, we used a very novel gene-editing technique called CRISPR and tagged a gene that is very strongly implicated in Parkinson’s disease called alpha-synuclein so that we can monitor the gene life. What happens in Parkinson’s disease is this particular gene is up-regulated. The expression of this protein goes up in the case of Parkinson’s disease, which leads to the subsequent degenerative process leading to the neuronal death as seen in Parkinson’s disease. So the overall aim of the Parkinson’s disease research lab is to monitor and modulate the levels of alpha-synuclein so that it does not reach that level of toxicity. For that we need a tool to screen for drugs that can actually lower the levels of alpha-synuclein in a human patient. The research that we did in this particular paper, we have modeled the increased alpha-synuclein levels and tagged it using the CRISPR technique with a luminescent light emitting gene which is called Nanoluc or Nanoluciferase so that whenever that protein is produced in the cell it will glow, it will emit light. So the ultimate target of this kind of work is how to reduce the light that has been produced. What we essentially did if I tell you in very brief steps; we put that light producing gene behind this alpha-synuclein which is important for Parkinson’s disease. Then we tried to create a situation where that particular protein is going up, up-regulated. And we want to see whether more and more light is getting emitted. That’s what we did. Next step is to screen for drugs which can actually bring that light production down, implying the efficacy of a screened drug. So that is the whole aim of a project like this.
So what would be the level of toxicity that you would consider being that borderline dangerous level that you would have?
Basu: That’s a very good question. It actually depends on various factors; it is not only the level but beyond a certain threshold this particular protein actually starts clumping together and it forms aggregate-like structures. Now these aggregated or the clumped protein that’s alpha-synuclein in a clumped or aggregated state is believed to be toxic. So at what point it will exactly start clumping, that is still being researched extensively. There are several factors which can be implicated to this aggregation process but the overall thing is when this protein starts aggregating it can becomes toxic to neurons and kill them eventually as seen in Parkinson’s disease.
The tool that you’re developing is screening those drugs to see if they’re bringing that level down?
Basu: Yes. Let’s look into an example. Say, in a normal human being there are ten molecules of alpha-synuclein protein. In Parkinson’s disease that number goes up to maybe one hundred. When it goes up in numbers it actually facilitates this process of aggregation as described earlier. So this number going up versus the aggregation process is very strongly related. What we essentially are doing is we developed a tool to actually see when that number is going up from ten to one hundred so we can monitor that number and eventually prevent it from going up. When that kind of a thing happens there comes into place the efficacy of the drug which will be screened in future studies which can retain that number to ten so that it doesn’t go up. If it goes up it will create problems later.
The aggregation what would that look like in the humans, does that produce more symptoms, how does that look?
Basu: It actually forms physical aggregates in cells. Consider a number of proteins, alpha-synuclein being one of the principle components of that aggregate, but there are some other proteins also which can contribute to the process. So it’s actually a round-shaped aggregated clumped protein structure that is seen in these neurons which are called the dopaminergic neurons that degenerate during Parkinson’s disease. What it looks like; there are several scientific techniques to actually visualize that. One of them is called a staining technique immunohistochemistry where they probe for a particular protein and we have seen that in Parkinson’s disease the propensity of forming aggregate is tremendously increased compared to a normal healthy human. So that’s the basic difference, but you can actually visualize the aggregates. And scientifically these aggregates are called Lewy bodies. It’s a very classical feature or let’s say a pathological feature of Parkinson’s disease. But what is the problem with a brain disorder like this? You cannot monitor something like this inside your brain whether it’s forming aggregates or not. These studies can only be performed after a person has actually passed away with the disease. All the research that we are doing is using these postmortem human brain samples which are from either control samples (healthy individuals)or Parkinson patient samples.. I would think this is actually a very tiny step that we have taken but it is actually maybe a very important platform in the future for screening drugs.
You mentioned the measuring of light, how crucial is that in your research?
Basu: That is exactly our research because normally that particular gene does not have any light activity in any cell type. It will be normally produced, synthesized and the protein that is being generated from that gene will do its role. But the beauty of a gene-editing technique like CRISPR is that we can genome edit very efficiently and very effectively. Using this technique we have put the light producing gene (Nanoluc) behind alpha-synuclein, which you don’t see under normal circumstances. We have engineered these cells so that whenever alpha-synuclein is produced in the cell it actually glows light. It actually produces light which is called luminescence scientifically and we measured that light production. Whenever we measure light we know that alpha-synuclein is being synthesized in the cell. And as I already mentioned the whole point of this project is to engineer a cell where alpha-synuclein is tagged with a light producing gene. This would help at a later stage to screen fordrugs to keep the light production in its basic level.
For people that are not familiar with this measuring of the light system can you describe or help them visualize how that measuring is done?
Basu: There are certain natural creatures that we get in the environment like for instance fireflies. We all know fireflies and how a firefly glows in the dark. What helps them glow in the dark? It is a particular protein which emits light and that is actually seen in the darkness. Similarly, there are some deep sea shrimp which produce light-producing proteins in them. The protein that we have used in this particular study to engineer the alpha-synuclein gene is actually derived from deep sea shrimp; it has its natural capacity to glow when provided with its substrate. But when I’m saying is glowing does not mean that it’s like a light. You have to measure that light produced in a scientific setting but you can definitely do that. This is what you call a luminescence; it’s a form of light production. And we have specific instruments that can actually detect luminescence. Whenever we add a substrate to it; these proteins, these are actually enzymes, convert the substrate and they produce lights so that can actually be measured through different kinds of instruments that are available in scientific settings.
Are there any screening tools like what you’re doing right now out there or is this something that’s brand new?
Basu: The novelty of this work actually I would say is twofold. Firstly, the technique by itself where we have implemented a system like CRISPR to study a human disease like Parkinson’s is novel. We don’t have any research that’s done before. And the second thing is that, the particular gene that we are studying alpha-synuclein; there are no studies reported earlier where a light producing gene has actually been tagged efficiently behind alpha-synuclein. So looking into the broader aspect of this paper, although I’m narrowing it down to Parkinson’s disease, but it has actually tremendous application to even some other diseases. Any disease let’s say; any gene which is strongly implicated in any disease. For example, a gene called beta-amyloid which is very strongly implicated in Alzheimer’s disease can also be tagged using this same way. That will actually facilitate the process of real-time monitoring of that particular gene. So we have actually proposed two things or done two things for this paper. One is a novel idea where we are actually proposing that many of these genes can actually be targeted and be used in this similar way. And second we have actually taken an example of this because we are a Parkinson’s disease research lab and we have shown that this process is implemented successfully.
What implication does you research have for future treatment or future screenings?
Basu: We are a basic research lab right. So what we believe is the technique and the tool that we have created has actually created a platform for screening of novel drugs in the future. And we expect that some drug which can be very effective in Parkinson’s disease by bringing the levels of alpha-synuclein can be validated very easily and screened very easily through a system like this. So to answer your question directly; this system has a lot of indirect reference to Parkinson’s disease patients. My system will not be doing anything directly to the patient but definitely it is going to help them by providing a screening platform for different kinds of drugs which can be efficient for Parkinson’s disease.
How did you yourself get involved in this research?
Basu: I did my undergrad in India and once I moved to the United States I really didn’t have a very clear vision what I want to pursue for my PhD. I kept myself open to exploring different avenues of biomedical research. And this school actually provides a fantastic opportunity to rotate in different labs to see which area of research you really want to pursue. This was one of my rotation labs and once I rotated I made up my mind that I’m definitely going to work on this. And keeping in mind the novelty, the projects, the atmosphere; I thought that this would be the best choice for me to pursue my PhD. Gradually over the years my interest in Parkinson’s disease grew. Not only Parkinson’s but other forms of neurodegenerative diseases as well, like for instance Alzheimer’s.
Is there any particular reason for the focus on the neurological diseases?
Basu: It just intrigues me, how this process works. Every researcher will say there are intrigued by a process. But I don’t know, I somehow got attracted to the field of neurodegenerative disease. I cannot think of any specific reason.
If you could describe what is CRISPR and how that impacts this research.
Basu: CRISPR is actually a gene-editing technique; it helps to edit genes. Every cell of our body has DNA, which produces RNA, which produces protein in turn. This is the central dogma of molecular biology. It is very hard to manipulate the DNA of a cell. By manipulating; I’m trying to mean editing DNA , as in deleting portions of it, introducing some other sequences.. All these are different forms of editing that can be done on the DNA. CRISPR is actually a very, very simple system that originated in bacteria. It’s actually a bacterial defensive mechanism against virus. There are three major lead researchers who thought that if bacteria can so effectively protect themselves from virus by editing their genome how about if we put this system in human cells. Can we achieve the same thing that was the question that they asked. And they did it very, very effectively. So it’s a very simple two object thing that, when delivered effectively in the human cells can efficiently edit the genome. In our study what we have done is, we have edited the genome by inserting this Nanoluciferase gene right behind alpha-synuclein. That’s how we made use of technique like CRISPR and that’s the beauty of the system that you can actually edit the genes very easily. Now with the editing comes in ethical grounds also, right? Essentially if people misuse this particular gene editing technique, they can create super humans (literally), they can create babies with super intelligence. Because all the genes responsible for proper development if they can edit all the bad things out of it, you’re essentially going to create a super human right? So there comes the ethical part of the whole technique. And there’s a lot of debate going on around the world as to what extent can you actually apply CRISPR. For us we are actually using this technique to modify a disease-related gene. So that’s definitely well within that ethical boundary I would say.
Is there anything that I didn’t ask you that you would like to add?
Basu: Not really that I can think of. In most of our research we have definite multiple steps that we need to take in the future as to understand where we are heading to using this particular tool. But we are very hopeful that at some point of time this tool might be very effective for drug screening and for the treatment of Parkinson’s disease.
What was your undergrad in?
Basu: My undergrad was in biotechnology.
END OF INTERVIEW
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If you would like more information, please contact:
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subhrangshu.guhathakurta@ucf.edu
Christin Senior
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