Parkinson's disease (PD), the second most prevalent neurodegenerative disorder, is characterized by progressive loss of dopaminergic neurons in the substantia nigra. Although the molecular mechanisms of PD remain incompletely understood, mitochondrial dysfunction has emerged as a central pathological driver, highlighting the urgent need for therapies targeting mitochondrial homeostasis. In this study, we demonstrate that rhynchophylline (Rhy), a bioactive alkaloid from Uncaria species, exerts neuroprotective effects by restoring mitochondrial dynamics. Thermal proteome profiling identified dihydrolipoamide acetyltransferase (DLAT) as a direct target of Rhy. Genetic ablation of DLAT induced mitochondrial fragmentation and abolished Rhy-mediated beneficial effects on mitochondrial structure and function. Mechanically, Rhy binds to the N-terminal lipoyl domain of DLAT, allosterically disrupting its interaction with sirtuin 4 (SIRT4) and subsequently enhancing DLAT lipoylation, a critical post-translational modification for mitochondrial energy metabolism. In vivo, Rhy administration ameliorated motor deficits and dopaminergic neurodegeneration in both the 6-OHDA-induced and A53T α-synuclein transgenic PD mouse models. Single-nucleus RNA sequencing further highlighted the clinical relevance of DLAT dysregulation in PD. Collectively, our findings establish Rhy as a promising PD therapeutic candidate and delineate DLAT as a pivotal node in therapeutic targets by promoting mitochondrial fusion and bioenergetics, offering a novel mechanistic avenue for neuroprotection.

Rhynchophylline  /  DLAT  /  Lipoylation  /  Mitochondrial dynamics  /  Parkinson's disease  /  Neuroprotection  /  Target identification  /  Thermal proteome profiling