Urolithin improves α-synuclein aggregation and DNMT1 expression in rotenone model of Parkinson’s disease

α-synuclein aggregation is a key hallmark of Parkinson’s disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). We examined the multi-targeting effects of urolithin (UA, UB, UC, UD, UE, UM5, and UM6) against α-synuclein aggregation using an in-silico and in-vitro approach. For in-silico analysis, several potential targets were selected like 1XQ8 (α-synuclein monomer), 1H1D (catechol-o-methyltransferase), 2BK3 (monoamine oxidase-B), 3IAM (NADH dehydrogenase), 4I5I (Sirtuin-1), and 5WVO [DNA methyltransferase-1], which play key role in α-synuclein aggregation, levodopa degradation, and mitochondrial dysfunction. In protein-protein docking analysis, 5HF9 (acetylcholinesterase, AChE) was found to interact with 1XQ8 dimer, forming a more stable complex with two additional H-bonds and one salt bridge, which indicates AChE's role as a nucleator in α-synuclein aggregation. In ligand docking and molecular dynamic studies, urolithin-A (UA) formed a more stable complex with 1XQ8, 4I5I, and 5WVO compared to specific inhibitor 1XQ8-ZPD2 and specific activator 4I5I-resveratrol. While other urolithins (UE, UM5, UC, and UD) displayed a more stable complex with 5HF9, 2BK3, 1H1D, and 3IAM compared to specific inhibitor 5HF9-physostigmine, 2BK3-selegiline, 1H1D-BIA, and specific activator 3IAM-resveratrol complexes, respectively. The blood-brain barrier permeability of UA (QPlogBB: −0.97) was predicted to be more than levodopa (QPlogBB: −1.44) and less than rotenone (QPlogBB: 0.08). DNMT1 inhibitor (5-Aza-dC) and rotenone robustly decreased the DNMT1 and α-synuclein expression in Neuro 2 A cells which was significantly reversed by UA treatment at 31.25 µM concentration. These findings indicate the potential of urolithins, specifically UA, UC, UD, UE, and UM5 against α-synuclein aggregation.