Rho‐kinase inhibition by fasudil modulates pre‐synaptic vesicle dynamics

• Published in: Journal of neurochemistry Vol. 157; no. 4; pp. 1052 - 1068
• Main Authors: Saal, Kim Ann, Warth Pérez Arias, Carmina, Roser, Anna‐Elisa, Christoph Koch, Jan, Bähr, Mathias, Rizzoli, Silvio O., Lingor, Paul
• Format: Journal Article
• Language: English
• Published: England Blackwell Publishing Ltd 01.05.2021
• Subjects: Depletion; fasudil; Fluorescence; Hippocampus; Kinases; Neurodegeneration; recycling; Regeneration; Rho-associated kinase; ROCK; Rocks; Signal transduction; Stimulated emission; Stimulation; synapse; Synaptic transmission; synaptic vesicle; Synaptic vesicles; Vesicles; Western blotting; recycling; ROCK; synapse; fasudil; synaptic vesicle
• ISSN: 0022-3042; 1471-4159
• DOI: 10.1111/jnc.15274

The Rho kinase (ROCK) signaling pathway is an attractive therapeutic target in neurodegeneration since it has been linked to the prevention of neuronal death and neurite regeneration. The isoquinoline derivative fasudil is a potent ROCK inhibitor, which is already approved for chronic clinical treatment in humans. However, the effects of chronic fasudil treatments on neuronal function are still unknown. We analyzed here chronic fasudil treatment in primary rat hippocampal cultures. Neurons were stimulated with 20 Hz field stimulation and we investigated pre‐synaptic mechanisms and parameters regulating synaptic transmission after fasudil treatment by super resolution stimulated emission depletion (STED) microscopy, live‐cell fluorescence imaging, and western blotting. Fasudil did not affect basic synaptic function or the amount of several synaptic proteins, but it altered the chronic dynamics of the synaptic vesicles. Fasudil reduced the proportion of the actively recycling vesicles, and shortened the vesicle lifetime, resulting overall in a reduction of the synaptic response upon stimulation. We conclude that fasudil does not alter synaptic structure, accelerates vesicle turnover, and decreases the number of released vesicles. This broadens the known spectrum of effects of this drug, and suggests new potential clinical uses. Chronic treatment with the Rho kinase (ROCK) inhibitor fasudil did not affect basic synaptic protein abundance, but altered synaptic vesicles dynamics. In stimulated cultures, fasudil reduced synapsin1 phosphorylation and the size of the recycling pool. We propose that over‐active vesicles thus are degraded faster reducing the synaptic response upon stimulation. This broadens the spectrum of fasudil effects suggesting new clinical uses of this drug.