Why Should I Care About How Scientists Do Their Science?

Hi again everyone! Holidays had me completely out of the creative zone, but I’m back to tell you about the science of mental disorders. However, before that, I am going to start with a more broad post about how scientists find out about the brain.

Science seeks to understand how the universe works (in all its aspects), but to do so it also needs to invest in designing new tools that let us see things that were invisible before. With each advance, science encounters new obstacles that it must overcome to see things that are too small, too big or too complicated to assess with what is available at the time. Then the new knowledge reveals new obstacles at the same time that it poses a solution for old ones.

What is achievable in a lab grows exponentially with each new technique. The possibilities are increased not just by that single novel technique, which would already be great, but by the combination of that technique with all of the other old and new techniques. Additionally, researchers don’t only use the technique as it was originally thought, but push the boundaries of what can be done with it, too. We can now do things we would never have dreamed of when I took my first breath 23 years ago.

I have selected three of the most recent advances to show some of the most fancy things we can do in biology: gene editing with CRISPR/Cas (changing the DNA sequence), optogenetics (controlling neuronal activity with light) and tissue clearing (making organs and even whole mice transparent).

Gene editing

Gene editing basically means changing our DNA by design. We have to remember that the finishing date for the sequencing (i.e. spelling) of the human genome was only 16 years ago, and that science usually is quite slow, so that the fact that we can not only of read but actually change the genome is, to me, absolutely mind-boggling. Changing the genome allows us to control what happens in a cell: we can shut off genes to study their function (what goes wrong in a cell or an organism when there is something missing helps us understand what the gene does when it is present), or reverse mutations that cause disease to see if the disease is then cured. We could change the genome already before CRISPR/Cas was ready for application. However, it wasn’t until its arrival that this became doable by any lab, increasing the potential of genetic studies and bridging the gap (to an extent) between rich and poor labs.

CRISPR/Cas is a system that was discovered in bacteria. It could be said that it is the immune system of these basic organisms, as they use it to protect themselves from viruses (phages) that infect them. In bacteria, CRISPR/Cas works as follows: when a phage infects bacteria, they start copying their genetic material to create more viruses. However, the bacteria can take fragments of the phage's DNA and store them in their own genetic material as a memorandum that they were previously infected by that type of virus. The bacteria then copy segments of the phage's genome periodically. These copied fragments can travel around the cell and bind to the original phage genome, so that when the phage infects the bacteria again, the bacteria will be prepared to fight back. The duplex formed by the new phage's genome and the copied fragment from the bacteria can be recognized by the Cas protein that can then cut them, thus, preventing the phage from making more copies of itself and probably killing the bacteria in the process. Researchers have taken advantage of this to design their own fragments that can bind to specific sites in the DNA of cells. Then Cas would recognize this site and cut the cellular DNA. When the cell tries to repair the cut, it sometimes introduces a change there instead of keeping the original sequence. These changes can be innocuous to the cell's functioning, but sometimes they can inactivate the gene or make the protein derived from it useless for the cell.

If researchers introduce into the cell an additional (different) DNA fragment, the cell might even integrate the new fragment at the cutting point. Essentially, this means that we can inactivate a gene, but also add new ones to the genome. This has unimaginable potential in the generation of new models of disease that previously were extremely complicated to get. In other words, it makes the first step to understanding a disease (having something to study it on) faster and easier.

However, that’s not everything CRISPR/Cas can do. It could also be applied to humans in the future to treat genetic diseases that have no permanent cure without it. A genetic disease is an illness caused by an error in our genes. Genetic diseases can be monogenic—caused by a single change in one gene—or polygenic—caused by changes in several genes. CRISPR/Cas can be useful in the treatment of monogenic diseases, as we can easily target just one gene with this system. For example, CRISPR/Cas could be used to treat retinitis pigmentosa, a disease that causes (for now) incurable blindness. It could also be used to treat Duchenne Muscular Dystrophy (DMD), where the muscles of young boys weaken to such an extent, that their average life-expectancy is up to their late teens or early twenties. There is as of yet no known cure. However, restoring the gene to its healthy version with CRISPR/Cas in both diseases could prove to be effective.

Nonetheless, we must be cautious. You might have heard of the baby Chinese twins, Lulu and Nana, which were born in late 2018 after genetic modification to "prevent" HIV infection. It was later revealed that the treatment was unnecessary, as only the father was affected by HIV and transmission from father to children is unlikely. Additionally, even though the treatment reduced the risk of HIV infection, it might increase the vulnerability of the twins to other diseases. Although it might seem that the treatment was beneficial, we must take into account that we still do not know about all the risks that CRISPR/Cas therapy might entail. We do know that it is not always 100% accurate, and parts of the genome that were not originally targeted might be modified, with unpredictable consequences for the patients. There are still many obstacles to make CRISPR/Cas treatment safe and ethically permissible. Therefore, we will still have to wait for some years until its use on "whole" humans is allowed. For now, only therapies that can be applied to a group of cells (for example cancerous cells or bone marrow blood cell progenitors) that are afterwards re-inserted into the patient are in clinical trials (being tested in humans). New versions of the CRISPR/Cas system are being developed to decrease the toxicity of the therapy, for example by eliminating the cutting activity of Cas and enabling it to suppress gene expression in other ways.

If you want to know more about this, you can listen to the following episode of Science Focus podcast (by the BBC):

Optogenetics

As I just said, we can add new genes to a cell using genome editing tools. One example is the second technique I will explain now: optogenetics. To be able to use this technique, researchers first need to include certain proteins that react to light, called opsins, into the mammalian cells. They are proteins that activate when hit by light instead of chemical substances and they are especially interesting for research involving neurons. Neurons communicate with each other by passing on electrical current, i.e. when one signals to another to activate, the second neurons lets through a positive current before going to a stable state again. The positive current then starts to make changes in the neuron in reaction to the signal from the previous neuron and also decides if it should signal to the next one or not. If opsins are introduced into the neurons, they can let in a positive or negative current (depending on the type of opsin) and this then activates or inhibits the neuron, respectively. Because this effect depends on light, the researcher can decide when and where to shine the light (or express the opsin) so that the effects on the neurons are really closely controlled.

Due to the advances in scientific techniques, researchers can express opsins in specific subtypes of neurons or brain subregions and study those neurons and their connections without so much interference from other brain parts. This increases the specificity of research immensely. Essentially, we can know what happens when we activate one particular region of the brain, i.e. what each brain region is for, a question that has been at the centre of neuroscience research for decades. Unfortunately, such subtle manipulations weren’t available before, and researchers had to settle for patients with lesions to parts of the brain, or reproduce, quite broadly, the lesions in animal models.

The fact that we are able to very specifically activate parts of the brain and record the behaviour of an animal afterwards is already amazing enough. But this technique (again) might not only be limited to the lab, but could also be moved to the clinic.

For example, treatment of retinitis pigmentosa is also being considered by delivering opsins into the cells of the eye. However, as with CRISPR/Cas, there are still many issues to be circumvented before moving onto treatment of diseases of the brain, first of all, safe delivery of light to the cells that are affected by disease. In the case of the eyes this is not too difficult. However, if we think of treating brain disorders with this strategy, researchers need to come up with a way of activating the opsins without major surgery. Some ideas (like designing opsins that are sensitive to infrared light, which can go through tissues without needing to open the skull) are already in development, but it will be many years until these options are made available as standard treatment in hospitals (remember—researchers have the duty of finding treatment for patients, but it must be the safest possible treatment, of course).

On a side note, both CRISPR/Cas and optogenetics are clear examples of why basic research should be funded. If it weren’t for the knowledge obtained from scientists in basic research, working on organisms so far from us as bacteria, we would have never developed these techniques. Imagine the potential for research and for treatment that would have been lost. Next time someone tells you that basic science is not useful because the main goal of science is to be applied to help humans, cite these examples to them ;)

Brain clearing

The last technique I am going to introduce today is maybe the most impressive, at least with regards to pictures. It is called tissue clearing, and as the name says, it consists of clearing tissue, i.e. making it transparent. What is the use of just making something transparent? We can see through it, but what is the point if what we want to see is actually in the tissue, as is the case in research? Because we don’t only make it transparent, we also have specific cell groups emitting light! Then we can look through the microscope and see very deep into the tissue without having to cut it and destroy the structure. And take very nice pictures too, of course, like the one at the beginning, which was part of my MSc thesis.

This technique has been an amazing asset to neuroscience, especially. Because before we had to cut the tissue to see the neurons under the microscope, it was really complicated to determine connections between different structures in the brain, another one of the main questions in this area of research. However, now, combining brain clearing with the fluorescence of the neurons we can connect the dots much more easily.

But neurons don’t naturally emit light, so how do we make them do it? We can insert a gene that will give rise to a fluorescent protein using CRISPR/Cas! Furthermore, we can insert it under the control of a gene that is only expressed in a particular subset of neurons, so that only those will be fluorescent and we will be able to clearly trace the connection from those neurons to other brain structures. The same principle can be applied to the expression of opsins, so that they are only present in certain types of cells. There are actually other combinations that can make the neurons emit light at certain time points, or only if the cell has gone through certain conditions, giving us much more information than just its position and connections in the brain! However, this would take quite a long paragraph to explain and I think I already gave you an overdose of information today. If you are intrigued and want to know more, let me know in the comments section! :)

But why am I telling you all of this? For a simple reason: you should have an educated say in what will happen now. Especially in the case of CRISPR/Cas and optogenetics, society as a whole has the right and the obligation to generate a debate about what regulations should be in place. One of the most pressing issues currently is the modification of the germline (in embryos or the sperm or eggs of the parents). Modifying these will mean that the descendants of that family will carry whatever mutations are in place without having a say in it. Is that fair? What kind of risks might that have for the new babies? Here are some links to gain more understanding on the ethical implications of gene editing in babies.

Lay people can also tip the balance of the debate; however, it is no good starting a debate with people that are un- or misinformed. To take up a position you must know about the topic. And that is what I tried to do here. This is just the first step; to know more you can check out the references below and the links embedded in the text or try Google (but be sure that the sites you choose are trustworthy).

If you liked the post or you think more people should know about what the future therapeutics might be, be sure to share the post on social media, I will be eternally grateful :)

You can follow me on twitter @addictscience or on facebook at Laura Sotillos Elliott, to get news of my new posts.

References

Chial, H. (2008) DNA sequencing technologies key to the Human Genome Project. Nature Education 1(1):219

Science Focus podcast, BBC. https://www.sciencefocus.com/the-human-body/is-gene-editing-inspiring-or-terrifying-nessa-carey/

Daley, G. Q., Lovell-Badge, R. & Steffann, J. After the Storm — A Responsible Path for Genome Editing. N. Engl. J. Med. 380, 897–899 (2019).

Tian, X. et al. CRISPR/Cas9 – An evolving biological tool kit for cancer biology and oncology. npj Precis. Oncol. 3, (2019).

Pickar-Oliver, A. & Gersbach, C. A. The next generation of CRISPR–Cas technologies and applications. Nature Reviews Molecular Cell Biology (2019). doi:10.1038/s41580-019-0131-5

Wiegert, J. S., Mahn, M., Prigge, M., Printz, Y. & Yizhar, O. Silencing Neurons: Tools, Applications, and Experimental Constraints. Neuron 95, 504–529 (2017).

Shirai, F. & Hayashi-Takagi, A. Optogenetics: Applications in psychiatric research. Psychiatry Clin. Neurosci. 71, 363–372 (2017).

Adamantidis, A. et al. Optogenetics: 10 years of microbial opsins in neuroscience. Nature Neuroscience 18, 1213–1225 (2015).

Lewis, P. M., Thomson, R. H., Rosenfeld, J. V. & Fitzgerald, P. B. Brain Neuromodulation Techniques: A Review. Neuroscientist 22, 406–421 (2016).

Tainaka, K., Kuno, A., Kubota, S. I., Murakami, T. & Ueda, H. R. Chemical Principles in Tissue Clearing and Staining Protocols for Whole-Body Cell Profiling. Annu. Rev. Cell Dev. Biol. (2016). doi:10.1146/annurev-cellbio-111315-125001

Chung, K. & Deisseroth, K. CLARITY for mapping the nervous system. Nat. Methods 10, 508–513 (2013).

Gradinaru, V., Treweek, J., Overton, K. & Deisseroth, K. Hydrogel-Tissue Chemistry: Principles and Applications. Annu. Rev. (2018). doi:10.1146/annurev-biophys