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

Arun Pal,Andreas Hermann,Julia Japtok,Ashutosh Dhingra,Masin Abo-Rady,Hannes Glaβ,Banaja P Dash,Nicole Kreiter,Marcel Naumann,Tobias M Böckers,Peter Heutink,Benedikt Kretner,Jared Sterneckert,René Günther

doi:10.26508/lsa.202000764

Abstract

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.

Highlights

Amyotrophic lateral sclerosis (ALS, Table 1) is a devastating, incurable motoneuron (MN) disease
All induced pluripotent stem cell (iPSC) lines were matured to spinal motor neurons (MNs) in microfluidic chambers (MFCs), in which only axons could reach and fully penetrate the microgroove barrier of channels from the proximal soma seeding site to distal exits (Naumann et al, 2018) (Fig 1A), thereby enabling axon-specific studies with defined antero- versus retrograde orientation
We sought to combine loss of C9ORF72 functions (LOFs) of C9ORF72 with HREmediated gain of toxic functions (GOFs) in a meaningful manner with no overexpression artifacts to clarify the role of both debated mechanisms (Waite et al, 2014; Walker et al, 2017; Frick et al, 2018; Nihei et al, 2020)

Summary

Introduction

Amyotrophic lateral sclerosis (ALS, Table 1) is a devastating, incurable motoneuron (MN) disease. This diversity of affected genes and mutation types seems to contradict the common scheme of MN degeneration and the final clinical outcome in ALS and calls for a thorough, comprehensive dissection in clinically relevant models to reveal mutation-specific upstream versus more common downstream mechanisms during the progression of neurodegeneration To this end, we are using fast multichannel live imaging on compartmentalized axons in vitro at standardized distal versus proximal readout sites (Naumann et al, 2018) owing to the hotly debated role of membrane trafficking defects in many neurodegenerative diseases (Sheetz et al, 1998; Salinas et al, 2008; Veleri et al, 2018). We have previously reported about deficient mitochondrial and lysosomal organelle trafficking in iPSC-derived spinal motor neurons from ALS patients bearing mutant fused in sarcoma (FUS) (Naumann et al, 2018) and TDP43 (Kreiter et al, 2018), two frequent genetic causes of ALS

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