SLITRK2 knockdown rescues axonal and functional neuromuscular junction deficits in human iPSC-derived mutant FUS-ALS motor neurons

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by motor neuron (MN) loss caused by early axonal and synaptic changes. However, the mechanisms underlying these changes remain incompletely understood. Here, we exploit FUS-ALS to interrogate cell compartment-specific changes in RNA expression and regulation in human induced pluripotent stem cell (iPSC)-derived MN.

This approach identifies compartment-specific 3’ untranslated region (3’UTR) isoform expression in human healthy control MN axons. Further, it unveils altered 3’ UTR isoforms in ALS in different cellular compartments in genes such as SNCA, previously linked to neurodegeneration. Expression of SLITRK2 is most strongly and consistently upregulated across multiple FUS-ALS models, and its knockdown normalizes axonal defects linked to FUS-ALS.

Using a fully iPSC-derived neuromuscular microfluidics protocol, we show that SLITRK2 knockdown in FUS-ALS MN rescues neuromuscular junction (NMJ) signaling without affecting NMJ innervation or MN hypo-excitability. Our study provides insight into compartment-specific 3’UTR expression in ALS, presents a robust iNMJ-on-a-chip model, and links elevated SLITRK2 to axonal and synaptic defects in FUS-ALS. We thank Danielle Vonk for iPSC culture support, Svetlana Pasteuning-Vuhman for MN culture support, Youri Adolfs, Jacqueline Sluijs and Yana Verresen for technical support and Didier Trono for providing reagents.

This project was financially supported by Stichting ALS Nederland (TOTALS, ALS-on-a-Chip, ATAXALS, MUSALS, GoALS), the MAXOMOD and INTEGRALS consortia (E-Rare-3, the ERANet for Research on Rare Diseases), the ALS CURE project, Alzheimer Nederland, the EU joint Programs Neurodegenerative Diseases (JPND; TRIAGE to R.J.P.) (JPND, project 733051105 to W.W.M.P.), the PPP Allowance made available by Health-Holland, TopSector LifeSciences and Health, to the Prinses Beatrix Spierfonds to stimulate public–private partnerships (project LSHM17075, LSHM19015, LSHM20011, and HH-PPS-24076- PC to W.W.M.P.) and by the National Growthfund projects NXTGEN Hightech (to W.W.M.P.) and Ombion (to W.W.M.P. and R.J.P.), the Netherlands Organization of Scientific Research (VI.Veni.202.113 to M.K.), and the FWO (Research Foundation-Flanders, G027725N to S.D.C.) and the Muscular Dystrophy Association (MDA, 962700 to S.D.C.). Present address: Department of Biochemistry and Biophysics, Stockholm University & Department of Cell and Molecular Biology, Karolinska Institutet, Biomedicum, Stockholm, Sweden Present address: Center for Neurogenomics and Cognitive Research, Department of Functional Genomics, Vrije Universiteit Amsterdam, Amsterdam, The Netherlands These authors contributed equally: Rianne de Jongh, Anna F. Department of Translational Neuroscience, University Medical Center Utrecht Brain Center, Utrecht University, Utrecht, The Netherlands Rianne de Jongh, Anna F.

Wiersema, Charlotte H. van Dijk, Roos C. Smulders, Onur Basak, Jelle van Os, Sofie Peeters, Lauri M. Jeroen Pasterkamp VIB-KU Leuven Center for Neuroscience, Department of Neurosciences, KU Leuven, Leuven Brain Institute, Leuven, Belgium Laura Olmedo-Martínez & Sandrine Da Cruz Cell Biology, Neurobiology and Biophysics, Department of Biology, Faculty of Science, Utrecht University, Utrecht, The Netherlands Department of Pediatrics, Department of Clinical Genetics, Center for Lysosomal and Metabolic Diseases, Erasmus MC University Medical Center, Rotterdam, The Netherlands Alessandro Iuliano & W.

Pim Pijnappel The authors declare no competing interests. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.