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Using Reconstituted Stress Granules to Gain Insight into the Molecular Pathology of Neurodegenerative Diseases

Periodic Reporting for period 1 - Stress Granules (Using Reconstituted Stress Granules to Gain Insight into the Molecular Pathology of Neurodegenerative Diseases)

Reporting period: 2019-02-01 to 2021-01-31

Membrane-less compartments were recently discovered as an organising principle inside the cell. They were shown to form by phase separation, a mechanism well understood by physics but new to biology. Since this field of research is still young, the biochemistry and physiology of membrane-less compartments is not well studied. Importantly, membrane-less compartments were implicated in many diseases. Therefore, a better understanding of their biology and pathology could open up new avenues for treatment options.
The overall objective of the project was to gain a deeper mechanistic insight into two different types of membrane-less compartments: cytoplasmic stress granules (SGs) and nuclear transcription factor (TF) condensates. The goal was to study their properties in vitro and to understand what functional consequences their manipulation has in vivo.
I reconstituted minimal SGs in the test tube and studied the recruitment of different components that can also be found inside cellular SGs. I discovered that protein-protein interactions play a smaller role than previously thought and that RNA-protein interactions are likely to be the determining factor for localisation. Furthermore, SG properties can be affected by targeting the recruited proteins with small molecules rather than the components essential for their formation. This is important knowledge in order to design drugs that delay SG solidification and thus positively affect the onset of neurodegeneration.
Furthermore, I also formed TF condensates on DNA. We discovered that a different physical phenomenon leads to the condensation of TFs than to the formation of SGs. We could also explain, for the first time, how TFs can recognise certain functional regions on the DNA and how condensation makes the process of gene activation more robust to varying protein concentrations. This knowledge is essential if we want to control the condensation of TFs and thus gene activation in the future. Possible applications concern treating diseases such as neurodegeneration, cancer, or developmental defects.
In the first part of the project, the SG protein G3BP1 and RNA were used to form minimal SGs. The resulting compartments were biophysically characterised and partitioning of client proteins was studied. I discovered that the partitioning of some client proteins depends on the type of RNA used in the minimal SGs. This suggests that recruitment is at least partially determined by the RNA binding preference and that protein-protein interactions play a less important role than previously thought. This data was part of a manuscript published in the journal Cell which was accompanied by a press release describing the findings to the general public.
The metabolite lipoamide had previously been shown to slow down SG solidification in vivo and to affect the biophysical properties of FUS droplet in vitro. I used lipoamide to also study its effect on reconstituted minimal SGs. Even though G3BP1 is essential for SG formation and lipoamide affects ageing of SGs inside cells, it had no effect on G3BP1 droplets. However, it increased partitioning of FUS into the minimal SGs. This demonstrates that even though G3BP1 is essential for SG formation, their overall properties can be dictated by other components. Targeting those components can in turn change SG properties. This information is important to design treatment strategies against neurodegenerative diseases.
In the second part of the project, I reconstituted a different type of membrane-less compartment: TF condensates. For this, I purified the pioneer transcription factor (TF) Klf4 and analysed its condensation behaviour in the presence and absence of DNA. In collaboration with a biophysics and a theoretical physics group, we were able to show that Klf4 condenses on DNA at physiological concentrations using a mechanism that is different from phase separation. This was surprising but can explain several cellular observations that were previously puzzling.
In addition, we could show that Klf4 condensates localise to specific sequences on the DNA. We can now explain how TFs are able to distinguish functional regions from single binding sites that occur at other locations. Furthermore, we discovered that condensation is advantageous for correct localisation as compared to simple DNA adsorption: only in this way, TFs can robustly localise over a wide concentration range. This study was published on a pre-print server for early dissemination and is currently being revised for Nature Physics.
Both parts of the project give valuable insight into general condensate biology and are relevant for basic research that concerns membrane-less compartments. In addition, the data can be exploited by medically oriented researchers to design new treatment strategies against diseases that involve SGs and TF condensates. This includes neurodegenerative diseases, cancer, and developmental defects. Furthermore, we are in contact with Dewpoint Therapeutics, a start-up company that is screening small molecules for interference with liquid condensates.
The action has greatly increased our understanding of cellular condensate biophysics. Many such condensates, but in particular the two examples studied here, have been implicated in diseases. SGs can solidify and form cellular inclusions, causing neurodegeneration. So far, there are no treatment options available that target the cause of the disease and only limited options to slow down progression. The insights from this study can help design new drugs which have the potential to delay, or even prevent, solidification and thus neurodegeneration.
Furthermore, this work discovered a new mechanism of TF condensate formation and described novel concepts for the regulation of gene activation. With this knowledge, we are one step closer to be able to manipulate gene regulation at will. Dys-regulation of gene expression is a hallmark of many cancer but also plays a role in other diseases such as neurodegeneration and developmental defects. The potential long-term benefits of studies such as this one are thus widespread.
Stress Granules findings