Disrupted macrophage metabolic adaptation and function drive senescence-induced decline in vertebrate regeneration

Rationale: Senescent cells accumulate with age and contribute to impaired tissue regeneration. Here, we developed a senescence-accelerated zebrafish (SAZ) model, characterized by accelerated senescence-like traits and a significant impairment in caudal fin regeneration. Methods: To investigate the underlying mechanisms of this regenerative defect, we employed a multifaceted approach. We used transgenic zebrafish lines for 4-D tracking of macrophage subsets during regeneration and performed parabiosis to assess the impact of systemic factors. Then, we isolated macrophages by FACS-sorting for a comprehensive transcriptomic study using RT-qPCR, enabling us to analyze both senescence markers and metabolic markers specifically within SAZ macrophages. Furthermore, we conducted phagocytosis assays to evaluate macrophage function. To explore the role of specific metabolic pathways, we used pharmacological treatments with oligomycin and galloflavin. Results: Our findings revealed that the reduced regenerative potential in SAZ was partly attributable to an impaired macrophage response during regeneration. We observed higher expression of the senescence marker cdkn2a/b in SAZ macrophages, which correlated with their reduced ability to polarize into a pro-inflammatory phenotype and exert efficient phagocytosis. These observations were linked to a significant downregulation of ldha, a key enzyme in lactate production, specifically within SAZ macrophages at 24 hours post-amputation. Enhancing anaerobic glycolysis in the SAZ model during early regeneration restored ldha expression, normalized macrophage activation dynamics, and ultimately rescued caudal fin regeneration. This rescue was entirely abolished by co-treatment with galloflavin, a direct inhibitor of LDH isoforms A and B, thereby underscoring the critical role of lactate metabolism in the regenerative process. Conclusion: Collectively, our findings demonstrate that senescence impairs regeneration by altering macrophage metabolic adaptation and functions, providing novel insights into the interplay between aging and regenerative capacit