Rapid and automated quantification of TDP-43 and FUS mislocalisation for screening of frontotemporal dementia and amyotrophic lateral sclerosis gene variants

Background Identified genetic mutations cause 20% of frontotemporal dementia (FTD) and 5-10% of amyotrophic lateral sclerosis (ALS) cases: however, for the remainder of patients the origin of the disease is uncertain. The overlap in genetic, clinical and pathological presentation of FTD and ALS suggests these two diseases are related. Post-mortem, 97% of ALS and ∼50% of FTD patients show redistribution of the nuclear proteins TDP-43 or FUS to the cytoplasm within affected neurons. We exploited this predominant neuropathological feature to develop an automated method for the quantification of cytoplasmic TDP-43 and FUS in human cell lines. Results Utilising fluorescently-tagged cDNA constructs to identify cells of interest, the fluorescence intensity of TDP-43 or FUS was measured in the nucleus and cytoplasm of HEK293 and SH-SY5Y cells. Confocal microscope images were input into the freely available software CellProfiler, which was used to isolate and measure the two cellular compartments. Significant increases in the amount of cytoplasmic TDP-43 and FUS were detectable in cells expressing known ALS-causative TARDBP and FUS gene mutations. Pharmacological intervention with the apoptosis inducer staurosporine also induced measurable cytoplasmic mislocalisation of endogenous FUS. Additionally, this technique was able to detect the subtler effect of mutation in a secondary gene ( CYLD ) on endogenous TDP-43 localisation. Conclusions These findings validate this methodology as a novel in vitro technique for the quantification of TDP-43 or FUS mislocalisation that can be used to assess the pathogenicity of predicted FTD- or ALS-causative mutations.