Studying RNA binding protein function in eukaryotic mRNA processing and neurodegenerative diseases
RNA binding proteins regulate RNA processing, which is critical in the control of gene expression. The TAR DNA binding protein (TDP-43) is an essential RNA binding protein whose dysfunction and aggregation are tightly associated with neurodegenerative disorders like amyotrophic lateral sclerosis (ALS), or Lou Gehrig’s disease, and frontotemporal dementia (FTD).
Our lab studies the cellular function and molecular structure of RNA binding proteins, such as TDP-43, to understand fundamental mechanisms regulating these proteins and to contribute in the development of therapies and detection tools for associated neurodegenerative disorders.
TDP-43 Function and Aggregation
The main questions addressed in our work:
- How is TDP-43 homeostasis regulated under non-pathological conditions?
- What factors trigger TDP-43 aggregation?
- How does TDP-43 loss of function contribute to ALS and FTD pathogenesis?
We found a novel dual phosphorylation site in TDP-43, p-T153/Y155, which is activated in response to proteotoxic stress, i.e., heat shock response.
p-T153/Y155 is specifically phosphorylated by MEK, the central kinase in the MAPK/ERK pathway.
p-T153/Y155 is recruited to the nucleolar compartment under normal conditions, and is upregulated after heat shock.
Phosphorylation is not associated with aggregates, autophagy, or degradation. Instead, p-T153/Y155 remains soluble under conditions of proteotoxic stress.
Heat shock-induced phosphorylation of TAR DNA-binding Protein 43 (TDP-43) by MAPK/ERK kinase regulates TDP-43 function. Li W, et al., J. Biol. Chem. 292(12):5089-5100, 2017 (PMID: 28167528).
TDP-43 Self Assembly and Aggregation
TDP-43, similar to other RNA binding proteins linked to ALS and FTLD, self-assembles and binds other ribonucleoproteins to form RNA-protein rich granules in cells (e.g., nucleolus, Cajal bodies, stress granules, etc.).
Formation of ribonucleoprotein granules is mediated by liquid-liquid phase transitions as seen by the formation of liquid droplets using purified TDP-43. These complexes are aggregation prone and, under certain conditions, may form pathological inclusions.
TDP-43 Aggregation and Intracellular Propagation Assays
We study the process of aggregation using purified recombinant TDP-43. Aggregate complexes formed over time are analyzed by semi-denaturing SDS agarose gel electrophoresis (SDS-AGE) and immunoblotting.
Using these assays we found that:
- High molecular weight fibrillar aggregates are preceded by oligomeric complexes.
- ALS-linked mutations, A315T and M337V, dramatically accelerate aggregation.
Our cellular reporter for TDP-43 aggregation shows that rTDP-43 aggregates (labeled green) seed and propagate intracellular aggregates (labeled red). This propagation is also increased by the ALS-linked mutations as seen by counting the aggregate-positive cells at 48 hours or 6 days after treatment.
French RL, Reeb AR, Aligireddy H, et al. TDP-43 oligomers detected as initial intermediate species during aggregate formation. bioRxiv [preprint]. 2018. bioRxiv 499343; doi: https://doi.org/10.1101/49934.