2017 KdVS Drug Therapy Research Update

Location: 2017 Koolen-de Vries Syndrome Patient Advocacy Summit

Date: July 14, 2017

Katrin Linda, a PhD student researching at Radboud University Nijmegen, spoke about Koolen-de Vries Syndrome cellular and molecular research.

To more easily understand the role that the KANSL1 gene plays, Katrin presented a simplified scenario. Working efficiently is easier when working in a small, organized, and cleaned up environment. So, in our homes we clean up our trash/waste and once in a while a garbage truck passes by, collects our trash, takes it to a factory where the waste is burned or recycled and then the energy, as well as the recycled goods, come back to our home. If this “waste factory” was malfunctioning and the garbage trucks could no longer travel to our homes, then trash would not be recycled/reduced, the trash would pile up at our homes and ultimately, we would not receive the energy from the factory.

Now what does this have to do with KANSL1? All of our cells develop a similar system in order to keep our cells clean and working efficiently; this process is called autophagy. In fact, last year’s Medicine Nobel Prize winner was awarded to a gentleman who examined autophagy. We expect that KANSL1 is working in a complex that is important for the regulation of autophagy. This complex is called the nonspecific lethal complex, or NSL complex; and within this complex KANSL1 is really important to keep all of the proteins working together. Without KANSL1 this protein would not hold together. Within a cell the NSL complex is required to make many genes, including those required for autophagy, available for protein production: Our DNA strands are quite long and we need to develop a system in order to pack our DNA into the cells. This is done by wrapping the DNA around the histones, which are actually little proteins, and it packs it really tiny. When the genes are wrapped around the histones, they are not available for transcription; so, you cannot make the proteins out of these. In order to do so, the cells need to develop another system that make the genes (that are required at a certain moment) available for transcription; this is done by so-called histone modification for which the NSL complex is needed. When KANSL1 is not available, we assume that this protein complex cannot form and the genes that would be required cannot be expressed.

We have different models available to examine if the clean-up process in the cells has actually changed on the KANSL1 mutations: There is a KdVS mouse model present. We actually share 99% of our genes with mice! Researchers were able to manipulate the KANSL1 gene in the mouse to produce a Koolen-de Vries Syndrome mouse. To evaluate whether this KdVS mouse has a change in learning abilities, the mice are given object recognition tasks. Mice are very curious about anything that is new, so mice are put in a cage with two objects and the mice are allowed to explore both objects. Both objects are new, so the mice will explore both at the same time. Then in the second session, one of the objects is replaced with a new one, so if the mouse remembers which object it has already seen, it will explore the new object more thoroughly than the object it already saw. Using this test, an assumption can be made about whether the mouse has a shorter memory.

In the first session, the Koolen-de Vries Syndrome mice explored the objects about the same amount of time. In the second session (where one of the two objects were replaced), the KdVS mice did not seem to remember which object they’d already seen and they explored both objects for a similar amount of time. The control mice, ones without the KANSL1 mutation, were able to remember which object they had seen and explored the newer object more thoroughly in their second session. It was concluded that mice with the KANSL1 mutation, which represents individuals with both the mutation and the microdeletion, have impaired short-term memory; so, he mice show some features of Koolen-de Vries Syndrome patients.

Mouse models are very effective for testing symptoms like behavior as their genes are so close to the human genes. However, there is this 1% difference and when it comes to understanding detailed mechanisms and drug testing rodents are less suitable due to differences in brain structure and organization. The rodent’s brain will not 100% mimic human cell mechanisms and drug effects. Drugs that showed to be effective in mice sometimes do nothing to the human. Because of the differences in mice and humans, a human model was preferred.

To simplify the human model, human cells can be obtained, cultured in a dish, and then various drugs and different treatments can be applied to the cultured cells and observed to see if the cells can be returned closer to their normal function. This would result in a simplified human model, better understanding of disease mechanisms, and would allow for drug screenings that eventually would allow for better drug therapy.

Katrin Linda’s interest and research is in the brain and the brain cells. Obtaining post-mortem brain material is very rare and in order to do large drug screenings a large amount of cell material is required, so post-mortem material is not a good solution. In 2006, another Nobel prize winner offered us the solution of reprogramming fibroblasts, in this case, to stem cell-like cells, that can then be shaped into any cell that you want. So, by that, patient’s fibroblasts can be reprogramed into Induced Pluripotent Stem (IPS) cells, and then generate patient neurons. At the 2015 KdVS family gathering, fibroblasts (skin samples) were taken from some individuals with KdVS and from those fibroblast samples, IPS cells were made and neurons were able to be generated from them.

Using the waste truck example previously mentioned, it was seen that in the control samples all of the cell’s waste trucks were of similar sizes. The samples with the KANSL1 mutations had some very large waste trucks. From this example, we assume that the IPS cells (control) are working appropriately and cleaning up the waste effectively. In the patient samples, something is not working correctly – so the question is: Is the factory not working, or do we need to push the waste trucks a little bit to get more waste into the factory?

A drug was used to treat the cells for 10 minutes. The drug, an autophagy inducer, was used to help the trucks push forward to get the waste into the factory. The effect of this drug was that the waste trucks were even smaller than the control sample. Ideally, research will find a drug that causes the waste trucks in the KdVS patient samples to match the size of the waste trucks in the control sample. For example, let’s say the control waste trucks come to collect garbage every two weeks. If the patient sample’s trucks are collecting once per month, this would not be enough. However, if they come every day, this would also not be efficient. The aim is to find a drug that helps to create a balance.

Before doing the experiment with brain cells, the morphology of the cells was assessed. Often you can get an idea about the function of the cells and how they communicate by assessing their morphology (such as shape and appearance). If they have longer arms, the cells make more contacts and are more effective. If the cells have shorter arms, then they are less effective. From a morphology standpoint, the cells of the patients with KdVS did not have significant differences from the cells from the control patients. So, the question is can we push the neurons a bit forward? This is a part of the project that will be done in the near future. Jumping back to the waste truck analogy, let’s say we find a drug that helps the waste trucks to deliver correctly again. Then, the question becomes whether the cells will function normally. To test the cell function, microelectrode arrays are used. Cells are cultured in a small dish with small electrodes that can record electrical signaling incorporated. Neurons communicate with each other with electrical signaling and this communication between the neurons can be recorded. While the cells generate in the dish, the cells will actually form a network with each other.

By observing the recording, it’s possible to see that the neurons around one electrode will start to communicate and then a few seconds later, more neurons will start to communicate. As they generate, they will develop a network and communicate at the same time. Ideally, all the neurons will fire at the same time and the same distance. In the example presented during the session (not a KdVS patient), it was observed that the neurons had difficulty organizing the synchronization of events and had difficulty communicating with each other. During future research, the idea is that when we actually have a drug that helps us to balance the autophagy situation, the drug would be evaluated to see if it changes the development of the network.

Since April of 2017, one new PhD students have begun working on the Koolen-de Vries Syndrome research. The students will continue researching and will generate personalized patient neurons and look for array sequencing. The students will look at all of the genes that are expressed in the cells and compare them between patients. Hopefully, genes will be located that are either expressed higher or lower than the control sample. As an example, let’s say that the researcher finds that autophagy genes in these cells are higher expressed. The researcher can consult an existing drug database that includes information of drugs already tested on cells and their changes on gene expressions. A drug that is used to reduce autophagy gene expression (in order to get the waste trucks back to the normal size) will be chosen and then tested on the specific KdVS cells to see if there is improvement.