Targeting protein phase separation and aggregation in neurodegenerative TDP-43 proteinopathies

Neurodegenerative disorders represent an increasing threat to the world public health and economy. There are no drugs that can substantially slow or prevent disease progression. Our understanding of the molecular basis of these diseases thus urgently needs to be improved. Hence, we are committed to identify novel drug targets for possible development of more effective therapies. Majority of neurodegenerative disorders are characterised by proteinaceous aggregates (proteinopathies) that accumulate in either the extracellular milieu or intracellular compartments of the affected cells. Frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS) and to an increasing extent also Alzheimer’s disease (AD), are characterised by cytoplasmic aggregates of TDP-43, a nuclear RNA-binding protein (RBP). It is generally accepted that under stress conditions within the cells TDP-43, along with some other RBPs, other proteins and RNAs, coalesces into cytoplasmic membraneless organelles, the so-called stress granules (SG). SGs allow the cell to cope with stress by transiently stalling normal protein translation, moving towards synthesis of cytoprotective proteins, and restoring other protein translation again as stress passes and the SGs dissolve. But during chronic stress exposure, such as in ageing, when defects in normal cellular processes accumulate, TDP-43 may start forming irreversible and insoluble aggregates resulting in degeneration of neuronal cells. This raises one of the most important questions in the field - What drives nuclear protein TDP-43 to accumulate and aggregate in the cytoplasm? The answer most probably lies in the changes of TDP-43 de novo expression, modifications, localization and/or degradation that are all afflicted by changes in TDP-43 protein-protein interactions. This project will thus focus on changes of the TDP-43 interactome emerging as TDP-43 shifts from soluble to coalesced state within SGs upon oxidative stress and, conversely, SGs disassembly following removal of oxidative stress. Our aim is to identify and mechanistically/pathologically characterise stress-related protein interactors of TDP-43, to increase the understanding of neurodegenerative disease mechanisms in order to facilitate the search for therapeutic targets. To this end the proposed project has three objectives: 1. Determine transient changes in the interactome of TDP-43 following oxidative stress and return to normal state. 2. Determine disease relevance of stress-related interactors of TDP-43. 3. Gauge the therapeutic potential of modulating selected interactors on TDP-43 aggregation. We will be implementing cutting-edge procedures, such as APEX2 for the identification of dynamic in vivo protein-protein interactions, which will enable us taking instantaneous “interactome snapshots” of TDP-43 to determine changes in its interactome upon oxidative stress exposure and return to normal state. These will provide us with far more exact insight into molecular processes leading to aggregation of TDP-43. The PI and international collaborators have extensive experience in the molecular mechanisms of ALS and FTD to execute this quite complex and ambitious project. The PI has pioneered in a number of topics of the ALS and FTD research, by extensively characterizing the disease related mechanisms of TDP-43, FUS and other genes, especially with regards to their perturbed RNA biology, nuclear transport, turnover and pathology. Our group was also one of the first pointing to perturbed stress granule regulation in these diseases. Therefore, we are confident to successfully carry out the research presented in this proposal. In conclusion, we are confident that investment into this research will bring us closer to understanding the disease processes and facilitate the search for treatments of TDP-43-associated disorders and other similar proteinopathies.