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Subsequent projects

Investigations on TDP-43-function in sporadic ALS and familial ALS with TDP-43 mutations

ALS is a neurodegenerative disorder affecting motor neurons (MNs). Neuropathologically, it is characterized by cytoplasmic mislocalization and aggregation of the RNA-binding protein TDP-43. Although 90% of cases occur sporadically (sALS), 10% of ALS cases occur in a mendelian pattern of inheritance (familial ALS, fALS). Interestingly, in a subset of fALS cases mutations in TDP-43 can be found but it is unknown whether these mutations correspond functionally to the aggregations seen in sALS. Hence, we propose to investigate this in patient stem cell-derived MNs of fALS patients with TDP-43 mutations, sALS cases with an increased insolubility of TDP-43 and healthy controls. We will apply eCLIP-seq to identify changes in the RNA-binding pattern, RNA-seq to delineate sALS/fALS-specific transcriptome changes and APEX2-proximity labeling to elucidate the TDP-43 interactome in the different states.

 

Primary project: TDP-43 pathology and cellular phenotypes in genome-engineered and iPSC-derived neurons of patients with ALS

 

Final Report

In this project, we aimed to explore commonalities and differences in transcriptome patterns, specifically alternative splicing (AS), between sporadic (sALS) and familial amyotrophic lateral sclerosis (fALS) in induced pluripotent stem cell (iPSC)-derived motor neurons (MN). Our fALS cohort consisted of patients with pathogenic variants in TDP-43. In sALS cases, TDP-43 does not contain any pathogenic variants but becomes aggregated in cytoplasmic inclusions in MNs in late stages of the disease. TDP-43 is a RNA binding protein involved in AS. Hence, it is thought that the biochemical changes of TDP-43 occurring in ALS affect AS.

We performed RNA-sequencing of iPSC MN of sALS and fALS patients and compared them to healthy controls. We identified that in both ALS modalities, AS changes can be observed in iPSC MN, highlighting a joint molecular phenotype of different ALS cases. When comparing the individual AS events, we found a high correlation between AS changes in fALS and sALS, suggesting a common origin of AS in both forms of ALS. To investigate if TDP-43 RNA binding is associated with these AS changes, we conducted enhanced crosslinking and immunoprecipitation (eCLIP) sequencing of TDP-43 in iPSC MN from healthy controls. Our human MN eCLIP-seq of TDP-43 recapitulated the known TDP-43 binding modalities such a binding to UG-rich sequences and intronic RNA regions. Interestingly, when integrating this TDP-43 RNA binding data with the fALS and sALS AS data, we observed that a minor amount of AS events in ALS iPSC MN was bound by TDP-43, while a number of AS events did not exhibit TDP-43 binding. This suggests that in parallel to TDP-43 there may also be other AS factors involved in AS in ALS iPSC MN. We used mass spectrometry to identify other AS factors influencing the transcriptome in ALS iPSC MN and showed that these AS factors also contributed to AS in ALS iPSC MN.

Imprint

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