Image-based deep learning reveals the responses of human motor neurons to stress and ALS

SUMMARYAlthough morphological attributes of cells and their substructures are recognized readouts of physiological or pathophysiological states, these have been relatively understudied in amyotrophic lateral sclerosis (ALS) research. 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). Surprisingly, we reveal that previously unrecognized disease-relevant information is withheld in broadly used and often considered ‘generic’ biological markers of nuclei (DAPI) and neurons (βIII-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 analyzing MN cultures exposed to different extrinsic stressors, we show that heat stress recapitulates key aspects of ALS. 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 testing of biological hypotheses.