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Universidad de Sevilla research update – January 2017

We are delighted to share with you an research project update from Dr. Mario David Cordero Morales
from the Universidad de Sevilla. His project is called:

Evaluation of the autophagy process and the inflammasome complex as a possible therapeutic alternative for Tay-Sachs and Sandhoff diseases

This report shows the findings of the research financed by ACTAYS at the Seville University during its first year (2016) related to the pathogenic mechanisms of the Tay- Sachs and Sandhoff disease taken from skin fibroblasts obtained from patients that are affected by either of the diseases.

Project Approach:

To reach our goals, we have set three different approaches:

  1. To study the physiopathology of the cell cleaning process (autophagy) as well as the activation of a new inflammatory process called inflammasome, and its modulation by different drugs with the aim of finding a possible therapy that could slow down the progress of the diseases.
  2. To evaluate the efficacy of the drugs that show better outcome in the treatment of the skin fibroblasts in a model of “Caenorabditis elegans”, a worm that measures 1 millimetre with an estimated life expectancy of 2 weeks, whose genome is well known and will allow us to develop models of human diseases.
  3. Those drugs evaluated in the human fibroblasts and in the worm, that present high efficacy will be tested in a mouse model provided by the team at Cambridge (Dr. Cox and Dr. Cachón-González).

Main achievements during 2016:

All the cell cultures have been set appropriately, with exception of the fibroblasts of the adult variants as their cell growth has been considerably slow. This has therefore created a problem with the establishment of these particular lines and we plan to repeat the skin biopsies in these patients so that we can set the adult lines again.

On the other hand, we have observed an imbalance in the redox equilibrium. It shows a high degree of free radicals of mitochondrial origin, as well as the increased metabolism in the mitochondria, that as a consequence produces a high level of the oxidative stress. There is also a deficit of energetic production, which entails a decrease in cellular growth. This altered metabolism causes a high production of other waste metabolites in patient’s cells, such as the lactic acid. This is a consequence of the activation of another metabolic route, the glycolysis.

On the autophagy route, we have observed interesting alterations in the molecular autophagy process. In the normal process, the waste of the cell is contained by the cell’s membrane. It stays in the cell and becomes part of the autophagosoma, which will ultimately join the lysosomes. This in turn contains digestive enzymes that will degrade the autophagosoma, removing the cellular waste.

In this research, we have been able to observe that there are high levels (above normal) of other degrading enzymes inside the lysosomes such as cathepsin B. This is toxic for the cell at high levels. We have also observed an important increase of this enzyme in our patients. This excess has caused what is known as permeability of the lysosomes, and as a consequence the cathepsin B exits the lysosomes causing damage in the cells as well as a low joining of the lysosomes with the autophagosomes (i.e. the digestives enzymes with the toxic waste.) On the other hand, the autophagy seems to try to balance that loss of union between the enzymes and waste (lysosomes and autophagosomes) increasing proteins in the final stage of the autophagy process. All of this leads us to think that there is a failure in the final steps of the process and not in its beginning.

Interestingly and in addition to this, we have noticed that the same process happens in the samples of the mice’s brains used in Cambridge. This gives us a perfect model for the trial of the drugs. As for the worm model, the protocol for the cultures and its maintenance has been set. There are two mutant strains identified, Hex-1 and Hex-2, that correspond to the loss of function of the human hexosaminidase, and therefore, Tay-Sachs and Sandhoff models. So at the beginning of 2017 we hope to have the worm cultures started.

Another outcomes (in human fibroblasts and in mice) is the apparent alteration, still t be confirmed, of the AMPK route. This is a master metabolism regulator that regulates the autophagy routes, cellular growth, the cellular metabolism, cellular death, etc. This discovery is important because there are molecules that induce its activation and therefore they could be candidates to test for a treatment. We hope to have final conclusions before summer.

However, due to the huge complexity of the diseases, we have started a massive approach to study a big set of molecular routes. To get a gene to express a final product (a protein, a hormone, etc), that gene must go through a process known as transcription, the step form DNA to RNA. The study of the transcriptors, known as transcriptomic, gives a great amount of information of the pathologic map of a disease, as well as a customized map of each patient. We have designed a study of the transcriptom for all the patients, as well as the mouse model at Cambridge. This includes not only more than 48.000 genes, but also new sequences of RNA as the microRNA (very little molecules), etc. This will allow us to make customized analysis for each patient, to see which routes are altered and therefore we could design possible drugs for treatment.

This part of the project has already started and the research fellow hired by ACTAYS has isolated the RNA of the mice which are already being analysed. We hope to have the data within the next month. The research fellow has also started the extraction of the RNA of the patients to study the human outcomes in parallel. The analysis of such an amount of data will take months. However, we hope to have a first report close to summer to deliver and present to the families.

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