Knocking out C9ORF72 Exacerbates Axonal Trafficking Defects Associated with Hexanucleotide Repeat Expansion and Reduces Levels of Heat Shock Proteins

• Published in: Stem cell reports Vol. 14; no. 3; pp. 390 - 405
• Main Authors: Abo-Rady, Masin, Kalmbach, Norman, Pal, Arun, Schludi, Carina, Janosch, Antje, Richter, Tanja, Freitag, Petra, Bickle, Marc, Kahlert, Anne-Karin, Petri, Susanne, Stefanov, Stefan, Glass, Hannes, Staege, Selma, Just, Walter, Bhatnagar, Rajat, Edbauer, Dieter, Hermann, Andreas, Wegner, Florian, Sterneckert, Jared L.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 10-03-2020; Elsevier
• Subjects: amyotrophic lateral sclerosis; Amyotrophic Lateral Sclerosis - genetics; Amyotrophic Lateral Sclerosis - pathology; Apoptosis - drug effects; axonal trafficking; Axons - drug effects; Axons - metabolism; Benzhydryl Compounds - pharmacology; C9ORF72; C9orf72 Protein - genetics; C9orf72 Protein - metabolism; Cell Differentiation - drug effects; Cytoplasmic Granules - drug effects; Cytoplasmic Granules - metabolism; disease modeling; DNA Repeat Expansion - genetics; Gain of Function Mutation - genetics; gene editing; Gene Knockout Techniques; heat shock proteins; HSP40; HSP40 Heat-Shock Proteins - metabolism; HSP70; HSP70 Heat-Shock Proteins - metabolism; Humans; induced pluripotent stem cells; Induced Pluripotent Stem Cells - drug effects; Induced Pluripotent Stem Cells - metabolism; Models, Biological; Motor Neurons - drug effects; Motor Neurons - metabolism; Motor Neurons - pathology; Nerve Degeneration - pathology; Pyrrolidinones - pharmacology; Transcriptome - genetics; HSP70; disease modeling; induced pluripotent stem cells; HSP40; C9ORF72; amyotrophic lateral sclerosis; gene editing; heat shock proteins; axonal trafficking
• ISSN: 2213-6711; 2213-6711
• DOI: 10.1016/j.stemcr.2020.01.010

In amyotrophic lateral sclerosis (ALS) motor neurons (MNs) undergo dying-back, where the distal axon degenerates before the soma. The hexanucleotide repeat expansion (HRE) in C9ORF72 is the most common genetic cause of ALS, but the mechanism of pathogenesis is largely unknown with both gain- and loss-of-function mechanisms being proposed. To better understand C9ORF72-ALS pathogenesis, we generated isogenic induced pluripotent stem cells. MNs with HRE in C9ORF72 showed decreased axonal trafficking compared with gene corrected MNs. However, knocking out C9ORF72 did not recapitulate these changes in MNs from healthy controls, suggesting a gain-of-function mechanism. In contrast, knocking out C9ORF72 in MNs with HRE exacerbated axonal trafficking defects and increased apoptosis as well as decreased levels of HSP70 and HSP40, and inhibition of HSPs exacerbated ALS phenotypes in MNs with HRE. Therefore, we propose that the HRE in C9ORF72 induces ALS pathogenesis via a combination of gain- and loss-of-function mechanisms.
•Axonal trafficking is disrupted in MNs with hexanucleotide repeat expansion (HRE)•C9ORF72 knockout (KO) exacerbated phenotypes in MNs with HRE•C9ORF72 KO reduced heat shock proteins in MNs with HRE•Inhibition of heat shock proteins exacerbated ALS phenotypes in MNs with HRE
Sterneckert and colleagues generated isogenic induced pluripotent stem cell lines and demonstrated that MNs with hexanucleotide repeat expansion (HRE) in C9ORF72 show reduced axonal trafficking, which is not recapitulated by knocking out C9ORF72 in MNs from healthy individuals. In contrast, knocking out C9ORF72 exacerbated phenotypes in MNs with HRE, suggesting that both gain- and loss-of-function mechanisms contribute to C9ORF72-ALS.