Lage Pita, LucíaRodríguez Pérez, Ana IsabelLabandeira García, José LuisDomínguez Meijide, Antonio2025-07-042025-07-042025Neurotherapeutics Volume 22, Issue 2, March 2025, e005441933-7213https://hdl.handle.net/10347/42392ROCK inhibitors such as fasudil protected against dopaminergic degeneration and other neurodegenerative processes in several experimental models through inhibition of neuroinflammation and activation of survival signaling pathways, and clinical trials have been initiated. More recently, fasudil has been suggested to inhibit α-synuclein aggregation. However, this is controversial, particularly if it is a consequence of direct binding of the fasudil molecule to α-synuclein. We studied the mechanisms involved in the effects of fasudil on α-synuclein aggregation using the α-synuclein-T/V5-synphilin-1 model. Molecule-molecule interactions were studied using real time quaking inducing conversion (RT-QuiC). Fasudil decreased the number of cells with inclusions and the size of inclusions in dopaminergic neurons and glial cells, and inhibited α-synuclein aggregation and microglial endocytosis of aggregates. These changes were not due to changes in α-synuclein protein expression or phosphorylation and were related to ROCK inhibition rather than direct interaction with α-synuclein, as confirmed with a second ROCK inhibitor (Y27632) and ROCK gene silencing. We observed that ROCK inhibition downregulates several factors that are known to promote α-synuclein aggregation such as NADPH-oxidase-derived oxidative stress, intracellular calcium increase, and α-synuclein endocytosis, and promotes autophagy. The present results support that fasudil is a useful drug against Parkinson's disease progression. In addition to other reported neuroprotective properties, fasudil inhibits α-synuclein aggregation and microglial endocytosis of aggregates, which enhances the microglial inflammatory response. The effects of fasudil are mostly related to ROCK inhibition, which we have shown using two structurally different ROCK inhibitors and knockdown data, and further supported by using RT-QuiC.eng© 2025 The Authors. Published by Elsevier Inc. on behalf of American Society for Experimental NeuroTherapeutics. This is an open access article under the CC BY-NC-ND licenseAttribution-NonCommercial-NoDerivatives 4.0 Internationalhttp://creativecommons.org/licenses/by-nc-nd/4.0/Alpha-synucleinAngiotensinNeurodegenerationNeuroinflammationParkinsonRho-kinasejournal article10.1016/j.neurot.2025.e005441878-7479open access