C9ORF72-derived dipeptide repeat proteins have emerged as the pathogenic cause of neurodegeneration in amyotrophic lateral sclerosis and frontotemporal dementia (C9-ALS/FTD). However, the mechanisms underlying their expression are not fully understood. Here, we demonstrate that ZNF598, the rate-limiting factor for ribosome-associated quality control (RQC), co-translationally titrates the expression of C9ORF72-derived poly(GR) protein. A Drosophila genetic screen identified key RQC factors as potent modifiers of poly(GR)-induced neurodegeneration. ZNF598 overexpression in human neuroblastoma cells inhibited the nuclear accumulation of poly(GR) protein and decreased its cytotoxicity, whereas ZNF598 deletion had opposing effects. Poly(GR)-encoding sequences in the reporter RNAs caused translational stalling and generated ribosome-associated translation products, sharing molecular signatures with canonical RQC substrates. Furthermore, ZNF598 and listerin 1, the RQC E3 ubiquitin-protein ligase, promoted poly(GR) degradation via the ubiquitin-proteasome pathway. An ALS-relevant ZNF598R69C mutant displayed loss-of-function effects on poly(GR) expression, as well as on general RQC. Moreover, RQC function was impaired in C9-ALS patient-derived neurons, whereas lentiviral overexpression of ZNF598 lowered their poly(GR) expression and suppressed proapoptotic caspase-3 activation. Taken together, we propose that an adaptive nature of the RQC-relevant ZNF598 activity allows the co-translational surveillance to cope with the atypical expression of pathogenic poly(GR) protein, thereby acquiring a neuroprotective function in C9-ALS/FTD.

Nucleocytoplasmic transport (NCT) defects have been implicated in neurodegenerative diseases such as C9ORF72-associated amyotrophic lateral sclerosis and frontotemporal dementia (C9-ALS/FTD). Here, we identify a neuroprotective pathway of like-Sm protein 12 (LSM12) and exchange protein directly activated by cyclic AMP 1 (EPAC1) that sustains the nucleocytoplasmic RAN gradient and thereby suppresses NCT dysfunction by the C9ORF72-derived poly(glycine-arginine) protein. LSM12 depletion in human neuroblastoma cells aggravated poly(GR)-induced impairment of NCT and nuclear integrity while promoting the nuclear accumulation of poly(GR) granules. In fact, LSM12 posttranscriptionally up-regulated EPAC1 expression, whereas EPAC1 overexpression rescued the RAN gradient and NCT defects in LSM12-deleted cells. C9-ALS patient-derived neurons differentiated from induced pluripotent stem cells (C9-ALS iPSNs) displayed low expression of LSM12 and EPAC1. Lentiviral overexpression of LSM12 or EPAC1 indeed restored the RAN gradient, mitigated the pathogenic mislocalization of TDP-43, and suppressed caspase-3 activation for apoptosis in C9-ALS iPSNs. EPAC1 depletion biochemically dissociated RAN-importin β1 from the cytoplasmic nuclear pore complex, thereby dissipating the nucleocytoplasmic RAN gradient essential for NCT. These findings define the LSM12-EPAC1 pathway as an important suppressor of the NCT-related pathologies in C9-ALS/FTD.

Translating ribosomes accompany co-translational regulation of nascent polypeptide chains, including subcellular targeting, protein folding, and covalent modifications. Ribosome-associated quality control (RQC) is a co-translational surveillance mechanism triggered by ribosomal collisions, an indication of atypical translation. The ribosome-associated E3 ligase ZNF598 ubiquitinates small subunit proteins at the stalled ribosomes. A series of RQC factors are then recruited to dissociate and triage aberrant translation intermediates. Regulatory ribosomal stalling may occur on endogenous transcripts for quality gene expression, whereas ribosomal collisions are more globally induced by ribotoxic stressors such as translation inhibitors, ribotoxins, and UV radiation. The latter are sensed by ribosome-associated kinases GCN2 and ZAKα, activating integrated stress response (ISR) and ribotoxic stress response (RSR), respectively. Hierarchical crosstalks among RQC, ISR, and RSR pathways are readily detectable since the collided ribosome is their common substrate for activation. Given the strong implications of RQC factors in neuronal physiology and neurological disorders, the interplay between RQC and ribosome-associated stress signaling may sustain proteostasis, adaptively determine cell fate, and contribute to neural pathogenesis. The elucidation of underlying molecular principles in relevant human diseases should thus provide unexplored therapeutic opportunities. [BMB Reports 2021; 54(9): 439-450]