ChRmine Kinases, Computational Biology, and Everything in Between
The Molecular Biology of Optogenetics
What if I told you there’s an emerging technology being tested in labs right now that if commercialized could create revolutionary treatments for a variety of neurological, neurodegenerative, and psychiatric disorders? One which combines the use of light and genetic engineering in a new way to achieve a slightly futuristic seeming ability resembling modern day mind control, lies at the intersection of two (multi) billion dollar markets, and has already achieved crazy stuff such as giving sight to a person who was blind. It almost seems too good to be true, but it’s not. The key to this all was found in nature, in a place that seems super unexpected when you first hear it…
- What is Optogenetics?
- What are Opsins?
- What’s ChRmine?
- What I Did with this Information + What It Means
- Implications on the World and the Future
- Key Takeaways
What is Optogenetics?
Optogenetics is an emerging neuroscientific technique which uses light and genetic engineering to manipulate neurons in the brain (the thing I was just talking about!). This works because of a protein naturally found in unicellular algae called channelrhodopsin. Channelrhodopsin plays a big role in the photosynthesis of the plant, because when it detects the type of light it’s sensitive to (blue light), it signals to the plant to face the light and open its channels to begin the process of photosynthesis.
Scientists realized that they could isolate this protein, insert it into neurons in the brain, and then activate those neurons when they wanted to with the use of light. The way this works is that channelrhodopsin is injected into specific neurons in the brain, and a brain computer interface (BCI) which will shine light on those neurons is also put in the brain through stereotaxic surgery. Then, the light can be turned on at any point to activate the opsins and control the neurons either for excitation (increasing their activity) or inhibition (decreasing their activity) depending on the type of opsin being used. Channelrhodopsin is excitatory, but once the technique was discovered scientists were able to find other types of opsins that were sensitive to different colors of light and had different use cases to increase the abilities of optogenetics.
The reason optogenetics is so special in comparison to other preexisting brain stimulation techniques such as Transcranial Magnetic Stimulation (TMS) or Deep Brain Stimulation (DBS) is because it’s far more precise. Since only neurons which have been modified with opsins will react to light, scientists are able to stimulate very small groups or neurons and even single neurons at once without impacting all nearby neurons as well.
This entire operation is made possible thanks to channelrhodopsin and other types of opsins. Now you know what opsins do and why they’re significant to optogenetics. But what really are opsins anyways?
What are Opsins?
From a more biological standpoint opsins are plasmids, circular strands of DNA which can replicate and perform functions independently from a larger double helix structure of DNA like the shape typically associated with the term. They’re naturally occuring in some bacterial cells, and can provide unique abilities to people who possess them such as antibiotic resistance. Another key difference between plasmids and standard DNA structures is that standard structures are surrounded by a membrane called a nuclear envelope that separates it from the cytoplasm, but plasmids are naked DNA with nothing surrounding them.
At the next smallest level, plasmids are made of kinases. In this case since we’re looking at opsins which are proteins, the kinases are a specialized type called protein kinases. These are enzymes which accelerate chemical reactions and transform molecules called substrates into a different type called products.
Kinases are made of chains of amino acids, the compounds that make proteins. And finally, at the smallest level we’re getting into today, the amino acids are made up of pairs of organic molecules called nucleotides. There are two possible pairs of nucleotides: adenine and thymine or cytosine and guanine. Each pair can only go with its opposite, not itself or a nucleotide from the other pair. When a nucleotide is connected to its pair, they are called a base pair. There are three base pairs in every amino acid. At this level, these base pairs are the exact same thing that make up all living things, but for our purpose today we’re looking at them in terms of opsins. More specifically from now on, in terms of chRmine.
What’s ChRmine?
ChRmine (pronounced car-meen) is another type of opsin protein which is used for optogenetics. It’s similar to channelrhodopsin in that it’s used for the excitation of neurons, but different in that it’s sensitive to red light as opposed to blue light. Its main advantages as opposed to other opsins are that it has very good temporal resolution and does not require a lot of light to be activated. The parts of the brain where chRmine is best suited for and largely used in are the ventral tegmental area/VTA (used for reward, motivation, and cognition) and hippocampus (used for memory).
I chose to use chRmine for my project since as someone who does a lot of research on optogenetics, I look at resources that talk about channelrhodopsin all of the time. It was the first opsin discovered for optogenetics, and it works fairly well so this makes perfect sense. However, I was curious to learn about other types of opsins. So when I saw chRmine referenced on the Karl Deisseroth website, it piqued my interest and I decided to look into it for this project.
What I Did with this Information
In order to do everything that I did for this project (which I’ll get into in a minute), I used Benchling. Benchling is a cloud based informatics software that has lots of great features for projects in molecular biology. I started by looking at vectors of different subtypes of chRmine plasmids. For the beginning I only used one vector, which showed the pAAV-CaMKIIa-ChRmine-eYFP-WPRE plasmid. Before I did anything to it, it was just a black circle with pieces inside of different sizes and colors (the protein kinases). On the left hand side, Benchling showed the genetic code of the plasmid (in base pairs) sorted by protein kinase.
The protein kinase I was most curious about was the chRmine kinase, since that kinase is the namesake of the group of plasmids and the one kinase they must all have in common to belong to the group. Based on this intention, the first thing I did was isolate the chRmine kinase from the plasmid.
Once I did this, I translated its genetic code into amino acids, and pulled up a set of biochemical properties such as its molecular weight, the frequency of each type of amino acid within the kinase (there are 20 types), and the net charges based on acidity. I experimented with other features as well, but these were the features I found to be most interesting and helpful.
After I had performed certain functions to better understand one chRmine kinase as an individual, I began some work to see how they varied. To do this, I pulled up a different type of chRmine plasmid with as few shared kinases (other than chRmine) as possible. That plasmid is called pAAV-Ef1a-DIO-ChRmine-mScarlet-Kv2.1-WPRE, and my first step when using it was also to isolate the chRmine kinase from its plasmid.
Once I pulled up its isolated chRmine kinase, I noticed its genetic code was a totally different ordered sequence than that of the first kinase’s code. However, the two kinases still had the same number of bases/nucleotides, so I didn’t stop at just this observation. The next thing I did was set up an alignment between the two kinases, which essentially matches up nucleotides to see how many two given organisms have in common. This was when I discovered that not only did the two kinases have the same number of bases, but their quantities of each type of nucleotide were also identical.
So what does this mean?
What this means is that even though the two kinases work similarly from a general standpoint given that they’re still the same type of kinase and their properties fit into those of chRmine as a whole, they’re used for slightly different things in their respective plasmids.
This may not make a whole lot of sense at first, so here’s an analogy to think about it a little differently. In the english language, the word funeral has the same exact letters in it as the phrase “real fun”. Those two might both be English, but they have entirely different meanings! The same can be said with the kinases, but with genetic code instead of English. Their general premises may be the same but that’s not to say they can’t be used for different things.
Implications on the World and the Future
This technology still has a ways to go as it is in the mice trials stage. To be approved by the FDA and become commercialized, it needs to complete mice trials and then go through a series of human testing stages. However, this doesn’t take away from the fact that in the next 10 years this technology could change millions of lives. Just this year, optogenetics was used for the first time to give sight to a blind man. This is just one of the many unbelievable applications optogenetics has had and will have once it’s ready to be used on humans.
As for looking at chRmine specifically, since it has excitatory properties it has great applications for neurodegenerative conditions. Since chRmine typically targets the VTA and hippocampus which are used for cognition and memory, one neurodegenerative condition which this opsin is especially useful for targeting is Alzheimer’s.
The reason neurodegenerative conditions are called what they are is because over time as people age, neurons in a given part of their brain will degenerate in their abilities. Eventually, some neurons won’t work at all in the way they are supposed to. Specifically for Alzheimer’s, this exact process is occuring in the areas which chRmine is intended to target.
By performing optogenetics using chRmine on patients with Alzheimer’s or other forms of dementia, we can combat the adverse effects of the conditions. By forcing the neurons to consistently stay active, we can keep up the working state of neurons in this area which still do work, and bring back the abilities of degenerating neurons.
Not just this, but chRmine and other types of opsins as well have effective applications for other neurodegenerative conditions such as Parkinson’s, and even entirely different types of conditions such as anxiety and depression. People with brain based disorders who suffer all sorts of regular adversity will soon be able to seek effective and precise treatment in the form of optogenetics.
Many people still are hesitant to accept optogenetics as they’re concerned about the ethics of an invasive computer based brain implant. While this concern is certainly normal and a valid piece of the process of introducing new technologies with unconventional equipment such as invasive implants, society needs to move past these additional apprehensions in order to move forward and advance techniques which could have incredible applications. Plenty of people will be nervous about implementing this type of tool, but just as scientists will work towards finding less invasive applications consumers should also try to open up their mindsets to include the possibility of this technology in the way it currently exists. Especially in the context of medical applications.
Optogenetics has clearly shown that it has all around incredible potential, and that once it finally becomes available for human use it could have a multitude of life changing applications. This technology could be the future of neurology, and all that’s in its way now is a little bit more time.
Key Takeaways
- Optogenetics is an emerging neuroscientific technique which uses light and genetic engineering to manipulate neurons in the brain
- Optogenetics is able to be performed based on channelrhodopsin, a type of opsin protein which is naturally occuring in unicellular algae. Other types of opsins have since been discovered and can also be useful depending on what the user is trying to achieve
- Opsins are plasmids (independent circles of DNA) made of protein kinases, which are made of amino acids and at the smallest level made of nucleotides and base pairs (adenine, thymine, cytosine, and guanine)
- ChRmine is a type of opsin which is excitatory and activated by red light. It’s largely used in the VTA and hippocampus
- I used Benchling’s molecular biology features to isolate chRmine kinases from different plasmids, translate their bases to amino acids and evaluate their biochemical properties, and then compare their nucleotide sequences.
- I noticed that two chRmine kinases coming from very different plasmids had the same number of nucleotides and of each type of nucleotide, but in a different order. I discovered this was because they did slightly different things even if their general use case as parts of a chRmine plasmid were the same
- Although optogenetics is still a very much developing technology, it could have revolutionary applications in the future such as for people with Alzheimer’s and other forms of dementia
Thank you so much for reading my article, I hope you enjoyed it! My name is Raina Bornstein, I’m 15 years old, and I’m passionate about branches of neuroscience, neurological conditions, and biotechnology. I’d love to connect on LinkedIn, or you can reach out to me at rainabornstein@gmail.com to talk or collaborate. I can’t wait to hear from you!
