**Background:** Ageing is a major risk factor for cognitive decline and neurodegenerative diseases. Cellular senescence, a hallmark of ageing, occurs in post-mitotic neurons, particularly excitatory neurons (ENs), which are the predominant senescent cell type in the aged human brain. However, the molecular mechanisms driving EN senescence remain unknown. Growth differentiation factor 11 (GDF11) has been proposed as a systemic rejuvenation factor, but its role in neuronal senescence and brain ageing is unclear. This study investigates whether endogenous GDF11 in ENs regulates their senescence, brain ageing, and cognition.
**Methods:** The authors used immunofluorescence, immuno-electron microscopy, and quantitative PCR to assess GDF11 expression in the brains of adult mice (3 months old), marmosets (62 and 70 months old), and human cortical tissue (from 4 patients aged 23–60 years). To study GDF11 function, they generated a conditional knockout mouse line (GDF11f/f; CaMKIIα-Cre, termed GDF11cKO) to delete GDF11 specifically in post-mitotic ENs. In vitro, CRISPR/Cas9 was used to knock out GDF11 in Neuro-2a cells. Senescence was assessed by SA-β-Gal staining, transmission electron microscopy (TEM) for lipofuscin and mitochondrial morphology, and bulk RNA-seq. In vivo, focal AAV-mediated deletion of GDF11 in the cingulate gyrus 2 (Cg2) was performed in GDF11f/f mice. Electrophysiological recordings (whole-cell patch clamp) measured excitability and synaptic inputs. Dendritic morphology was analyzed via biocytin labeling and Sholl analysis. Cognitive function was evaluated using the three-chamber test (3CT) and novel object recognition test (NORT). Single-nucleus RNA-seq (snRNA-seq) of 24,803 nuclei from Cg2 was performed to identify transcriptional changes. Chromatin immunoprecipitation qPCR (ChIP-qPCR) assessed Smad2 binding to the p21 promoter. A double knockout mouse (CaMKIIα-Cre; GDF11f/f; p21f/f) was used to test p21 dependency.
**Key Results:** GDF11 was predominantly expressed in CaMKIIα+ ENs in the adult mouse, marmoset, and human brain (approximately 90% of GDF11+ neurons were CaMKIIα+). GDF11 mRNA and protein levels declined with age in mice (mRNA halved at 36 months vs. 3 months; protein reduced at 9 and 36 months). In GDF11cKO mice, SA-β-Gal+ senescent cells increased by over 50% in the insular, piriform, and cingulate cortices at 10 months, and the effect spread to the cingulate cortex by 17 months. Median lifespan was reduced from 25 months in GDF11f/f mice to 22.8 months in GDF11cKO mice (10% reduction). In vitro, GDF11 knockout in Neuro-2a cells doubled the proportion of SA-β-Gal+ cells (p<0.0001), increased nuclear area (p=0.0030), and caused lipofuscin accumulation (number doubled, p=0.0002; area increased, p=0.0274) and mitochondrial area reduction (p<0.0001). Bulk RNA-seq revealed 706 upregulated and 411 downregulated genes, with upregulation of p21 (Cdkn1a). In vivo, focal GDF11 deletion in Cg2 ENs caused hyperexcitability: action potential frequency increased (slope 0.30 vs. 0.18, p=0.000), input resistance increased (214 vs. 104 MΩ, p=0.000), and mIPSC frequency dramatically decreased (4.0 vs. 34.6 Hz, p=0.000). Dendritic morphology showed reduced apical dendrite length (2.83 vs. 3.77 mm, p=0.044) and spine density (1.61 vs. 6.28 per 10 μm, p=0.000), while mushroom spine diameter increased (0.80 vs. 0.66 μm, p=0.000). Behaviorally, fGDF11cKO mice showed impaired sociability (preference index S1-O lower, p=0.0118) and social memory (S2-S1 lower, p=0.0128) in the 3CT, and GDF11cKO mice failed to discriminate novel objects in NORT (discrimination index lower, p=0.0254). SnRNA-seq showed that GDF11 deletion upregulated p21 mRNA approximately fourfold and p53 approximately twofold in ENs. ChIP-qPCR confirmed increased Smad2 occupancy at the p21 promoter in GDF11KO cells (p<0.001). Double knockout of GDF11 and p21 rescued the increase in SA-β-Gal+ cells in the cingulate, insular, and piriform cortices at 17 months (p<0.0001 for rescue).
**Clinical Implications:** This study identifies GDF11 as an endogenous inhibitor of EN senescence and brain ageing, acting through the Smad2/3-p21 pathway. The findings suggest that maintaining or enhancing GDF11 signaling in excitatory neurons could be a therapeutic strategy to slow brain ageing, prevent cognitive decline, and potentially extend healthspan. The demonstration that GDF11 deletion shortens lifespan and impairs cognition in mice provides causal evidence linking neuronal senescence to organismal ageing. The conserved expression of GDF11 in human ENs supports translational relevance. However, the study is limited to animal models and in vitro systems; human validation and safety considerations for GDF11 modulation are needed. The region-specific effects (insular, piriform, cingulate cortices) may inform targeted interventions for age-related cognitive disorders.