Image‐based deep learning reveals the responses of human motor neurons to stress and VCP‐related ALS

AIMS: Although morphological attributes of cells and their substructures are recognised readouts of physiological or pathophysiological states, these have been relatively understudied in amyotrophic lateral sclerosis (ALS) research. METHODS: In this study, we integrate multichannel fluorescence high‐content microscopy data with deep learning imaging methods to reveal—directly from unsegmented images—novel neurite‐associated morphological perturbations associated with (ALS‐causing) VCP‐mutant human motor neurons (MNs). RESULTS: Surprisingly, we reveal that previously unrecognised disease‐relevant information is withheld in broadly used and often considered ‘generic’ biological markers of nuclei (DAPI) and neurons ( [Formula: see text] III‐tubulin). Additionally, we identify changes within the information content of ALS‐related RNA binding protein (RBP) immunofluorescence imaging that is captured in VCP‐mutant MN cultures. Furthermore, by analysing MN cultures exposed to different extrinsic stressors, we show that heat stress recapitulates key aspects of ALS. CONCLUSIONS: Our study therefore reveals disease‐relevant information contained in a range of both generic and more specific fluorescent markers and establishes the use of image‐based deep learning methods for rapid, automated and unbiased identification of biological hypotheses.