Protein thiol alterations drive pathologic liquid-liquid phase separation in the aging brain

Cellular homeostasis relies on the precise regulation of chemical processes, including protein posttranslational modifications (PTMs) and biomolecular condensation. Aging disrupts the equilibrium of these processes, increasing susceptibility to disease and mortality. Using advanced chemoproteomic techniques, we investigated cysteine PTMs in the aging mouse brain and generated web-based explorer (https://aging- redox-proteomics-viewer.streamlit.app/). Our findings reveal that age-related increases in thiol oxidation promote the formation of biomolecular condensates. In contrast, protein persulfidation, regulated by endogenous hydrogen sulfide (H2S) production, inhibits biomolecular condensation, thereby preserving protein function. These cysteine PTMs significantly alter the phase separation properties of synapsin 1, a key regulator of synaptic activity, contributing to impaired neurotransmitter release associated with aging and neurodegenerative diseases. Age-induced alterations in cysteine PTMs influence the phase separation properties of synapsin 1 and G3BP2, leading to impaired neurotransmitter release and defective stress-granule formation/resolution, respectively, both features associated with aging and neurodegenerative diseases. Mice deficient in cystathionine γ-lyase, the enzyme responsible for H2S production and protein persulfidation, exhibit reduced lifespans and spontaneously develop neurofibrillary tangles and protein aggregates with age. Our results highlight the therapeutic potential of protein persulfidation in reversing dysregulated biomolecular condensation, offering new avenues for using sulfide donors to mitigate age-related diseases.