Long road to neuron

“There is a great goal that we will achieve, of course, only in the long term, is the treatment of neurodegenerative diseases,” begins the story by Alexei Menzorov, a researcher at the Laboratory of Development Genetics ICIG SB RAS. “There are diseases in which a certain group of neurons suffer, and we would like to replace them.” Ideally, the picture looks like this: new cells are introduced into the patient’s brain that take on the functions of the dead. However, this is much easier to say than to do. Firstly, it is almost very difficult to introduce ready-made neurons and, most likely, they will not take root. Secondly, in order to introduce them, they must be taken from somewhere, and adult neurons, as you know, do not share.

Stem cells, and primarily embryonic stem cells (abbreviated ESC), are a special class of cells that have the ability to turn into cells of any tissues of the body. In the laboratory of development genetics, a whole class of work is carried out related to the ESC of the mouse. Although they pursue different tasks, they have a common problem.

“We plan to get neurons from ESC, enter their mouse into the brain and see if they will work there,” explains Alexey Menzorov. “But there is one problem-these neurons must somehow be marked.”

A common way to distinguish some cells from others is to integrate a special new area in the genome that encodes the formation of fluorescent protein. When irradiated with ultraviolet light, this protein will glow green or red. But if you just send a piece of such a protein-coding DNA inside the stem cell, then it will glow regardless of whether this cage will turn into neuron or into something else. Therefore, a system was developed in ICIG, which allows us to “include” the synthesis of protein only if the original stem cell either began to turn into a neuron or has already become it.

Although the set of genes in the living organism, in principle, the same for all tissues, cells of different species differ in which genes in them “work” and which proteins synthesize. For neurons, for example, such a marker was chosen a section of the genome, encoding the protein of the beta-tubul class III, and for cells-the neuron's predecessors, the protein of Netin was chosen.

The system, which was developed and used in ICIG, lies in the fact that DNA is initially cut in a certain place, then plasmid with the necessary sequence is introduced into the nucleus, which is built into the cut DNA. Then the DNA is glued, but now it turns out that with the synthesis of beta-tubulin or Nestin, the assembly and fluorescent markers proteins will turn on along with them. Thus, if ESC begins to turn into a neuron, it will begin to glow. If development goes along a different path, then there will be no fluorescence.

“We want - and we are moving to get neurons on the mouse model that we could select and monitor how they participate in the treatment of the damaged mouse brain,” explains Alexei Menzorov. “Now we have received ESC, which, firstly, in culture they themselves can turn into neurons and, secondly, from which we can get a transgenic mouse.”

The initial material is taken from the SPF-breaking of the institute. Researchers select mice embryos at an early stage of development, receive ESCs from them, introduce the desired section of the genome in them and receive cell culture. Then these cells are tested for “normality” - whether the number of chromosomes is correct. And then, when this check has been completed, you can already work with such cells.

One of the directions is to start turning them into neurons using certain substances. Further, these neural stem cells - that is, SK, which can only turn into neurons, but are not yet neurons - transplanted by experimental mice at the embryo stage or in the first days after birth. In adult mice, as the researcher clarifies, for this, you must first cause some damage to the brain - then there is a chance that the newly introduced SK will participate in the “repair”.

Another, no less interesting work, is a transplant of the “swept” ESC directly to the mouse embryo at the blastocyst stage, when the whole organism of the future mouse consists of only hundreds of cells. It turns out, according to Alexei Menzorov, “chimera”, “composite” mouse, since it develops not only from the cells of its parents, but also from the transplanted.

If the experiment goes normally, then the new swept cells begin to share and gradually turn into cells of different mouse tissues. Some of them will later turn into gametes - germ cells, and thus the offspring of this mouse will be transgenic, neurons will “glow” green in ultraviolet rays.
A luminous mouse, of course, is interesting in itself, but the purpose of this complex procedure is to get an animal whose cells contain a gene that can synthesize green fluorescent protein. Although it is actually present everywhere, it will “turn on” only in neurons.

Meanwhile, relatively recently it became known that not only ESCs can be turned into neurons, but, for example, skin cells - fibroblasts, if you influence the latter with certain genetic manipulations. However, how to check that a new cell is a neuron? The green fluorescent protein, the synthesis of which will start only in the neuron, will answer.

The ultimate goal of this large and painstaking work is to “see” what is happening with neurons, how they develop and how they react to a transplant to the mouse body. Studies are conducted in a large collaboration, together with the Novosibirsk State University and the Skolkovo Institute of Science and Technology.

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