Molecular mechanisms of α-syn abnormal phase separation in cognitive impairment induced by chronic intermittent hypoxia and the neuroprotective effects of Danshensu methyl ester.

The pathological association between α-synuclein (α-syn) deposition and neurodegeneration or cognitive dysfunction has been extensively studied for decades, but significant progress has yet to be achieved in clinical translation. Recently, the introduction of phase separation theory has provided a new perspective for elucidating the molecular mechanisms of α-syn deposition. We made the first discovery that chronic intermittent hypoxia (CIH) exposure induced fear memory deficits by damaging dopaminergic neurons in the ventral tegmental area (VTA). In vivo experiments showed that CIH exposure significantly increased α-syn expression and caused its abnormal deposition in the VTA, leading to neuron dysfunction. Furthermore, inhibiting α-syn expression effectively mitigated neuronal damage and fear memory deficits. Using an intermittent hypoxia (IH) model, we found that abnormal phase separation of α-syn mediates the transition from soluble state to fibrous state, which is the core mechanism of its deposition. α-syn phase separation is regulated by various factors, and we focused on reactive oxygen species (ROS), which were closely associated with α-syn liquid-liquid phase separation (LLPS) and notably altered in CIH/IH conditions. The results showed that CIH-induced ROS promoted the conversion of α-syn-EGFP phase separation droplets to fibrillar state. Moreover, in vitro experiments confirmed that hydrogen peroxide (H₂O₂) treatment induced abnormal phase separation of α-syn, while the antioxidant monomer drug Danshensu methyl ester (DME) effectively inhibited this process. Furthermore, in CIH and IH models, DME intervention significantly reduced ROS levels, inhibited α-syn abnormal phase separation, decreased intraneuronal α-syn deposition, and ultimately improved dopaminergic neuron damage and cognitive function. This study reveals that ROS-regulated abnormal phase separation of α-syn is a novel mechanism underlying CIH-induced neuron damage and might provide insights for the clinical treatment of related cognitive disorders.