The research team started with a clever comparative strategy. They used two well-known mouse strains with dramatically different fates as they age: C57BL/6J mice, which lose hearing rapidly, and CBA/CaJ mice, which retain hearing much longer. At nine months of age, auditory brainstem response testing confirmed what decades of research had suggested. C57 mice had significantly elevated hearing thresholds at all tested frequencies, from 8 to 32 kilohertz, compared with age-matched CBA mice. At 24 kilohertz, for instance, C57 mice required sounds roughly 35 decibels louder than CBA mice to elicit a detectable brainstem response.

With these functional differences established, the researchers sequenced the entire transcriptome of cochlear tissue from both strains. Principal component analysis revealed a clear molecular separation between the two groups, and differential expression analysis identified thousands of genes at different levels: 3,439 upregulated and 3,080 downregulated in the hearing-vulnerable C57 strain. But it was the pattern of enrichment that caught the team’s attention. Genes involved in ribosome assembly, translation and RNA metabolism were prominently altered, alongside inflammatory pathways such as NF-κB signalling and complement cascades. This convergence of translation-stress and inflammatory signatures hinted at a molecular bridge between cellular stress adaptation and inflammatory signalling in the degenerating cochlea.

That bridge, the researchers hypothesised, might be the stress granule. Stress granules are dynamic, membraneless condensates composed of RNA and proteins that assemble inside cells under diverse stress conditions. When cellular stress strikes, phosphorylation of a translation initiation factor called eIF2α halts protein synthesis, leaving a pool of non-translating mRNAs that cluster together with RNA-binding proteins like G3BP1 and TIA-1 through liquid-liquid phase separation. These granules protect transcript integrity and allow cells to reprogramme translation in response to stress. Once the stress passes, they rapidly disassemble. G3BP1 is the core nucleator of this process, making it a prime candidate for connecting stress responses to downstream inflammatory programs.

To test whether G3BP1 was genuinely tied to aging rather than strain-specific genetics, the researchers compared G3BP1 levels in young and aged mice of both strains. At two months of age, G3BP1 expression was comparable between C57 and CBA mice. But at nine months, G3BP1 protein levels were significantly higher in the cochleae of the hearing-vulnerable C57 mice than in age-matched CBA mice. This age-dependent emergence suggested that G3BP1 upregulation develops alongside strain-dependent susceptibility to hearing loss, rather than being a constitutive difference between the strains.

The team then turned to an in vitro model to dissect the mechanism. They exposed HEI-OC1 cells, a mouse cochlear sensory cell line, to hydrogen peroxide at 50 micromolar for 36 or 72 hours. This treatment induced a senescence-like phenotype, confirmed by increased senescence-associated β-galactosidase activity and elevated p21 protein expression. The senescence-associated secretory phenotype factors IL-6 and TNF-α were also significantly elevated at 72 hours. Crucially, G3BP1 expression rose alongside activation of the pyroptosis markers cleaved caspase-1 and GSDMD-N, the pore-forming fragment of gasdermin D that punches holes in cell membranes and triggers inflammatory cell rupture.

Pyroptosis is a lytic form of programmed cell death canonically linked to inflammasome activation. When the NLRP3 inflammasome assembles, it activates caspase-1, which processes inflammatory cytokines and cleaves gasdermin D. The N-terminal fragment of GSDMD then forms membrane pores, causing cellular swelling and rupture. In the cochlea, this pathway has been implicated in responses to oxidative stress, metabolic perturbation and sterile inflammation, all relevant to aging. Recent work has even shown that GSDMD-mediated pyroptosis contributes directly to cisplatin-induced hearing loss, with pharmacological inhibition or genetic deletion of GSDMD attenuating cochlear damage.

With the stage set, the researchers performed reciprocal loss- and gain-of-function experiments. When they silenced G3BP1 with siRNA and then exposed cells to hydrogen peroxide, levels of cleaved caspase-1 and GSDMD-N increased further, indicating that removing G3BP1 exacerbated caspase-1/GSDMD-mediated pyroptotic signalling. Conversely, when they overexpressed G3BP1 using a plasmid, levels of both pyroptosis markers dropped significantly at 72 hours. ELISA measurements confirmed that G3BP1 overexpression significantly reduced IL-18 release at both time points, though IL-1β was not significantly altered. Immunofluorescence analysis showed a clear tendency towards reduced ASC, the inflammasome adaptor protein, following G3BP1 overexpression, though the differences did not reach statistical significance.

These bidirectional findings support a protective role for G3BP1 against oxidative stress-induced pyroptosis-associated activation in cochlear cells. The researchers propose several mechanisms by which G3BP1 and stress granules might restrain inflammasome signalling. Stress granule formation has been reported to regulate factors like DDX3X, potentially limiting NLRP3 inflammasome assembly and downstream ASC speck formation. SG-associated stress responses may also influence mitochondrial ROS, mitochondrial DNA release or potassium efflux, all of which modulate inflammasome activation. However, the researchers caution that these mechanisms remain hypothetical in the context of age-related hearing loss and will require direct assessment of stress granule formation, DDX3X localisation and mitochondrial function in future studies.

The work arrives at a moment when inner-ear gene therapy is advancing rapidly. AAV-mediated base editing has recently achieved substantial and sustained auditory recovery in a mouse model of hereditary hearing loss, demonstrating that disease-relevant molecular pathways within the cochlea can be therapeutically manipulated. While hereditary hearing loss and age-related hearing loss differ fundamentally in aetiology, these advances highlight the therapeutic accessibility of cochlear molecular pathways. The researchers suggest that G3BP1 and stress granule-associated stress responses could represent potential targets for enhancing cochlear resilience during aging. They acknowledge important limitations: young animals were not included in the original sequencing experiment, a complete age-by-strain transcriptomic design is still needed, and in vivo gain- or loss-of-function studies will be required to determine whether G3BP1 directly modifies auditory decline during aging. Still, by identifying a stress-responsive regulator that connects chronic cochlear stress to inflammatory cell death, the study opens a new avenue for understanding and potentially treating one of aging’s most pervasive sensory deficits.

Subject of Research: Neuroscience

Article Title: Transcriptomics Implicates the G3BP1–SG Axis in Cochlear Hair‐Cell Pyroptosis During Age‐Related Hearing Loss

Article References: Li, C., Cao, Y., Zhou, J., Chen, Y., Tang, Q., Wang, S., Wu, Z., Wang, S., Zhang, Z., Xie, M., Huo, H., & Yang, H. (2026). Transcriptomics Implicates the G3BP1 – SG Axis in Cochlear Hair‐Cell Pyroptosis During Age‐Related Hearing Loss. Journal of Cellular and Molecular Medicine, 30(19), Article e71403. https://doi.org/10.1111/jcmm.71403

Image Credits: AI Generated

DOI: 10.1111/jcmm.71403

Keywords: Transcriptomics, Implicates, G3BP1, Axis, Cochlear, Hair, Cell, Pyroptosis, During, Related, Hearing, Loss