Concomitant gain and loss of function pathomechanisms in C9ORF72 amyotrophic lateral sclerosis.

Intronic hexanucleotide repeat expansions (HREs) in C9ORF72 are the most frequent genetic cause of amyotrophic lateral sclerosis, a devastating, incurable motoneuron (MN) disease. The mechanism by which HREs trigger pathogenesis remains elusive. The discovery of repeat-associated non-ATG (RAN) translation of dipeptide repeat proteins (DPRs) from HREs along with reduced exonic C9ORF72 expression suggests gain of toxic functions (GOFs) through DPRs versus loss of C9ORF72 functions (LOFs). Through multiparametric high-content (HC) live profiling in spinal MNs from induced pluripotent stem cells and comparison to mutant FUS and TDP43, we show that HRE C9ORF72 caused a distinct, later spatiotemporal appearance of mainly proximal axonal organelle motility deficits concomitant to augmented DNA double-strand breaks (DSBs), RNA foci, DPRs, and apoptosis. We show that both GOFs and LOFs were necessary to yield the overall C9ORF72 pathology. Increased RNA foci and DPRs concurred with onset of axon trafficking defects, DSBs, and cell death, although DSB induction itself did not phenocopy C9ORF72 mutants. Interestingly, the majority of LOF-specific DEGs were shared with HRE-mediated GOF DEGs. Finally, C9ORF72 LOF was sufficient-albeit to a smaller extent-to induce premature distal axonal trafficking deficits and increased DSBs.

High content organelle trafficking enables disease state profiling as powerful tool for disease modelling.

Neurodegenerative diseases pose a complex field with various neuronal subtypes and distinct differentially affected intra-neuronal compartments. Modelling of neurodegeneration requires faithful in vitro separation of axons and dendrites, their distal and proximal compartments as well as organelle tracking with defined retrograde versus anterograde directionality. We use microfluidic chambers to achieve compartmentalization and established high throughput live organelle imaging at standardized distal and proximal axonal readout sites in iPSC-derived spinal motor neuron cultures from human amyotrophic lateral sclerosis patients to study trafficking phenotypes of potential disease relevance. Our semi-automated pipeline of organelle tracking with FIJI and KNIME yields quantitative, multiparametric high content phenotypic signatures of organelle morphology and their trafficking in axons. We provide here the resultant large datasets to enable systemic signature interrogations for comprehensive and predictive disease modelling, mechanistic dissection and secondary hit validation (e.g. drug screens, genetic screens). Due to the nearly complete coverage of analysed motility events, our quantitative method yields a bias-free statistical power superior over common analyses of a handful of manual kymographs.