Objective:

To identify potential therapeutic strategies for motor neuron disease (MND) by identifying human genes that modulate TAR DNA-binding protein 43 (TDP-43) -mediated aggregation and toxicity.

Methods:

We conducted a pooled human genome-wide CRISPR activation screen in HEK293T cells expressing cytoplasmic-targeted TDP-43 protein mimicking pathology found in disease. This assessed the capability of over 18,000 genes to, when activated, modulate protein aggregation and confer cytoprotection in HEK293T cells with TDP-43 pathology. Top modulators of toxicity and TDP-43 aggregation were selected for investigation. Biochemical, microscopy and flow cytometry techniques were used to investigate the proposed influence top modulators have on TDP-43 pathology in vitro.

Results:

Subsets of genes were identified that when activated significantly protected from, or sensitised cells to, toxicity (601 ‘Protect’ genes; 733 ‘Sensitise’ genes. LFC > 1) and inhibited or enhanced TDP-43 aggregation (832 ‘Inhibit’ genes; 37 ‘Enhance’ genes. LFC > 1). This revealed that activation of a NF-KB signalling pathway modulator, which belongs to the leucine-rich repeat containing protein family, sensitised TDP-43 mutant cells to death. Further investigations confirmed that overexpression of this target sensitised cells to toxicity in the context of TDP-43 aggregation. Flow cytometry techniques revealed that target overexpression significantly increased the percentage of cells with insoluble aggregates (P=0.0196). Target knockdown using shRNA inhibited this effect on aggregation.

Conclusions:

This CRISPR activation screen identified hundreds of potential targets that modulate TDP-43 pathology. Manipulation of these target gene pathways may promote clearance or refolding of pathological TDP-43, supporting development of therapies for those living with MND.