LRRK2 kinase inhibition with MLi2 treatment

We inhibited LRRK2 kinase activity with the selective LRRK2 inhibitor MLi-2 (Tocris 5756). MLi-2 has low solubility in water, necessitating the use of a vehicle for solubilization. We chose to use the cyclodextrin Captisol® (Ligand RC-0C7-100) as a vehicle, due to its worldwide safety approval and use in human drug formulations (www.captisol.com/about). We bath sonicated 1 mg MLi-2 in 2 mL 45% Captisol®-PBS for ~ 2 h at room temperature (until complete solubilization of MLi-2). Solutions were filter sterilized prior to use. Primary cortical cultures were treated with 500 nM MLi-2 or Captisol®-only control (each 1 mL well treated with 0.4 uL stock in 100 mL fresh media; final Captisol® concentration 0.00016%) for 2 h prior to fixation, lysis, or electrophysiological recording. Treatment concentrations and times were selected based on the pharmacokinetic data collected by Fell and colleagues [50]. For western blot experiments in brain tissue, animals were injected intraperitoneally with MLi-2 or Captisol®-only control at a dose of 5 mg/kg, 2 h prior to rapid decapitation without anaesthesia. All tissues were collected, frozen, and stored as described above.