Divergent Molecular Pathways for Toxicity of Selected Mutant C9ORF72-derived Dipeptide Repeats

Abstract

Expansion of a hexanucleotide repeat in a noncoding region of the C9ORF72 gene is responsible for a significant fraction of Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD) cases, but mechanisms linking mutant gene products to neuronal toxicity remain debatable. Pathogenesis was proposed to involve the production of toxic RNA species and/or accumulation of toxic dipeptide repeats (DPRs) but distinguishing between these mechanisms has been challenging. In this study, we first use complementary model systems for analyzing pathogenesis in adult-onset neurodegenerative diseases to characterize the pathogenicity of DPRs produced by Repeat Associated Non-ATG translation of C9ORF72 in specific cellular compartments: isolated axoplasm and giant synapse from the squid. Results showed selective axonal and presynaptic toxicity of GP-DPRs, independent of associated RNA. These effects involved a MAPK signaling pathway that affects fast axonal transport and synaptic function, a pathogenic mechanism shared with other mutant proteins associated with familial ALS, like SOD1 and FUS. In primary cultured neurons, GP but not other DPRs promote the "dying-back" axonopathy seen in ALS. Interestingly, GR- and PR-DPRs, which had no effect on axonal transport or synaptic transmission, were found to disrupt the nuclear membrane, promoting "dying-forward" neuropathy. All C9-DPR-mediated toxic effects observed in these studies are independent of whether the corresponding mRNAs contained hexanucleotide repeats or alternative codons. Finally, C9ORF72 human tissues confirmed a close association between GP and active P38 in degenerating motor neurons as well as GR-associated nuclear damage in the cortex. Collectively, our studies establish compartment-specific toxic effects of C9-DPRs associated with degeneration, suggesting that two independent pathogenic mechanisms may contribute to disease heterogeneity and/or synergize on disease progression in C9ORF72 patients with ALS and/or FTD symptoms. Graphical AbstractActivation of protein kinases and inhibition of axonal transport, synaptic transmission, and nuclear structure are toxic effects common to unrelated FALS-related gene products. FALS-related mutant forms of SOD1 (mSOD1), FUS (mFUS), and C9-GP-DPRs (GP(n)) activate specific ASK1-MAPK pathway. Within axons, active ASK1-p38 pathway phosphorylates various substrates, including conventional kinesin, leading to the inhibition of fast axonal transport mediated by the translocation of this motor protein along microtubules. ASK1 can also inhibit synaptic transmission via JNK activation. Both pathways cause reductions in the availability of critical synaptic cargoes, synaptic dysfunction, and "dying-back" degeneration of neurons. On the other hand, C9- PR and GR-DPRs (PR(n) and GR(n)) activate other pathways, leading to aberrant alterations in nuclear structure and function and "dying-forward" degeneration of neurons, consistent with reports of transcriptional changes and activation of apoptosis in ALS. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/558663v2_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@162cbbeorg.highwire.dtl.DTLVardef@12e54caorg.highwire.dtl.DTLVardef@c0cdaborg.highwire.dtl.DTLVardef@1feb8d0_HPS_FORMAT_FIGEXP M_FIG C_FIG