Energy metabolism and membraneless organelles have been implicated in human diseases including neurodegeneration. How energy deficiency regulates ribonucleoprotein particles such as stress granules (SGs) is still unclear. Here we identified a unique type of granules induced by energy deficiency under physiological conditions and uncovered the mechanisms by which the dynamics of diverse stress-induced granules are regulated. Severe energy deficiency induced the rapid formation of energy deficiency-induced stress granules (eSGs) independently of eIF2α phosphorylation, whereas moderate energy deficiency delayed the clearance of conventional SGs. The formation of eSGs or the clearance of SGs was regulated by the mTOR-4EBP1-eIF4E pathway or eIF4A1, involving assembly of the eIF4F complex or RNA condensation, respectively. In neurons or brain organoids derived from patients carrying the C9orf72 repeat expansion associated with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), the eSG formation was enhanced, and the clearance of conventional SGs was impaired. These results reveal a critical role for intracellular energy in the regulation of diverse granules and suggest that disruptions in energy-controlled granule dynamics may contribute to the pathogenesis of relevant diseases.

Amyotrophic lateral sclerosis (ALS) is a devastating motor neuron disease. The molecular understanding of ALS is hampered by the lack of experimental models recapitulating disease heterogeneity and analytical framework integrating multi-omics datasets. Here, we developed a pipeline integrating machine learning and consensus clustering to analyze a large-scale dataset of patient-derived motor neuron models from Answer ALS. Compared to the transcriptome, proteomic profiling closely correlates with ALS pathology, which is interrogated to identify 110 proteomics-based biomarkers (Proteomics Markers for ALS 110, PMA110). Functional enrichment highlights dysregulation of ALS pathways, including protein translation and neuronal function. By integrating ALS subtype-specific proteins with patient postmortem proteomics, we found that RPS29 was consistently downregulated in ALS models and patient motor neurons. RPS29 is required for neuronal viability by maintaining ribosome profiling and accurate translation, and suppressing pathological translation. RPS29 downregulation suppresses translation of STMN2, an essential protein for motor neurons, in iPSC-derived motor neurons. Taken together, this study provides a robust framework for ALS proteomics, identifies RPS29 as a quality controller of protein translation, and presents a translational mechanism for STMN2 maintenance in ALS.

The RNA exosome plays a critical role in surveilling nuclear mRNA biogenesis and regulating co-translational mRNA decay in the cytoplasm. Unlike canonical translation, repeat-associated non-AUG (RAN) translation of a GGGGCC hexanucleotide repeat expansion (HRE) within an intron of the C9orf72 locus leads to the synthesis of neurotoxic dipeptide-repeat (DPR) proteins, contributing to the pathogenesis of frontotemporal dementia and amyotrophic lateral sclerosis (C9-ALS/FTD). However, it remains unclear whether aberrant RAN translation is monitored and regulated co-translationally or how C9orf72 HRE (C9-HRE) mRNA is degraded during this process. Here, we demonstrate that RAN translation triggers the rapid decay of C9-HRE mRNA. During this process, the RNA exosome engages the translating ribosome-C9-HRE mRNA complex to mediate RAN translation-coupled mRNA decay. Moreover, overexpression of EXOSC3, a key subunit of the RNA exosome cap, promotes RAN translation-coupled decay of C9-HRE mRNA and suppresses DPR production. In iPSC-derived neurons, a reduction in EXOSC3 levels blocks C9-HRE mRNA decay in a translation-dependent manner, further confirming its role in RAN translation surveillance. These findings highlight the essential function of the RNA exosome, particularly EXOSC3, in mitigating RAN translation-associated toxicity and preventing pathological DPR production. This work provides insights into potential therapeutic strategies for C9-ALS/FTD and may have broader implications for other disorders involving RAN translation.

Energy metabolism and membraneless organelles have been implicated in human diseases including neurodegeneration. How energy stress regulates ribonucleoprotein particles such as stress granules (SGs) is still unclear. Here we identified a unique type of granules formed under energy stress and uncovered the mechanisms by which the dynamics of diverse stress-induced granules are regulated. Severe energy stress induced the rapid formation of energy-associated stress granules (eSGs), whereas moderate energy stress delayed the clearance of conventional SGs. The formation of eSGs or the clearance of conventional SGs was regulated by the mTOR-4EBP1-eIF4E pathway or eIF4A1, involving eIF4F complex assembly or RNA condensation, respectively. In ALS patients’ neurons or cortical organoids, the eSG formation was enhanced, and conventional SG clearance was impaired. These results reveal a critical role for intracellular energy in the regulation of diverse granules and suggest that an imbalance in these dynamics may contribute to the pathogenesis of relevant diseases.