Mapping TDP-43 RNA binding partners in motor neuron differentiation and ALS pathology

TAR DNA Binding Protein 43 kDa (TDP-43) is an RNA/DNA-binding protein that is fundamentally implicated in the development and disease of the nervous system. It is predominantly localized in the nucleus, where it is implicated in multiple steps of transcriptional and post-transcriptional regulation. In particular, TDP-43 has a key role in alternative splicing, stability and transport of essential neurodevelopmental genes. Aberrant cytoplasmic mislocalization, ubiquitination, abnormal phosphorylation, cleavage and aggregation of TDP-43 are common histopathological hallmarks of a spectrum of neurodegenerative disorders, collectively known as TDP-43 proteinopathies, including amyotrophic lateral sclerosis (ALS). ALS is an age-related, incurable disease characterized by selective motor neuron (MN) degeneration. The progressive loss of upper and lower MNs in the brain, brainstem and spinal cord, cause paralysis and ultimately death. Aberrant TDP-43 mislocalization is found in 97% of all ALS cases, bridging both familial and sporadic forms of ALS. Given its pleiotropic nature and the multiple cellular processes that are aberrantly affected by TDP-43 aggregation and/or nuclear depletion, establishing direct causal links between TDP-43 and disease remains challenging. The molecular mechanisms by which TDP-43 mislocalization affects human motor neurons remains unresolved, as do its precise roles in normal motor neurogenesis.

Against this background, I wish to understand whether TDP-43 pathology is the result of a nuclear loss of function (e.g aberrant pre-mRNA processing), a cytoplasmic gain of toxic function, or a combination of the two. Specifically, I will address the following questions: (i) how does TDP-43 regulate motor neuron development; (ii) how and when does this regulation go awry in ALS (iii) and what target genes are affected; (iv) and how does TDP-43 post-translational modification influence its RNA-binding targets and splicing function? I will explore these questions in hiPSCs derived MNs from healthy and ALS donors generated in the Patani lab, combined with advanced transcriptomic and computational profiling approaches developed in the Luscombe group.

A major obstacle in addressing these questions is the lack of in vitro or in vivo models that recapitulate authentic ALS phenotypes in human motor neurons. Induced pluripotent stem cells (hiPSCs) provide an unprecedented opportunity to investigate the progression of molecular pathogenic events in a human setting that is conventionally inaccessible and therefore represents an ideal model to explore TDP-43 RNA targets in MN development and degeneration.
I will perform individual-nucleotide resolution UV-crosslinking and immunoprecipitation experiments (iCLIP) in the hiPSCs derived MNs to identify key TDP-43-RNA interactions over the course of MN differentiation in healthy and ALS-mutant MNs, and investigate conformation specific changes in TDP-43 binding targets. I will then computationally integrate iCLIP and RNA-seq data to uncover TDP-43-mediated alternative splicing, gene expression changes and alternative polyadenylation events. The analysis will reveal the link between binding sites and specific functional outcomes, and will guide downstream molecular investigations.

This work will expand our understanding of a fundamental protein in neurodegeneration, TDP-43. I will uncover the regulatory mechanism(s) of TDP-43 in MN differentiation and to capture early perturbations in its RNA binding targets, which may cause MN degeneration in ALS pathology. Ultimately, this work may provide key insight into molecular mechanisms and new therapeutic targets in ALS