Dysregulation of the AP2M1 phosphorylation cycle by LRRK2 impairs endocytosis and leads to dopaminergic neurodegeneration.

Mutations in the kinase LRRK2 and impaired endocytic trafficking are both implicated in the pathogenesis of Parkinson's disease (PD). Expression of the PD-associated LRRK2 mutant in mouse dopaminergic neurons was shown to disrupt clathrin-mediated endocytic trafficking. Here, we explored the molecular mechanism linking LRRK2 to endocytosis and found that LRRK2 bound to and phosphorylated the µ2 subunit of the adaptor protein AP2 (AP2M1), a core component of the clathrin-mediated endocytic machinery. Analysis of human SH-SY5Y cells and mouse neurons and tissues revealed that loss of LRRK2 abundance or kinase function resulted in decreased phosphorylation of AP2M1, which is required for the initial formation of clathrin-coated vesicles (CCVs). In contrast, overexpression of LRRK2 or expression of a Parkinson's disease-associated gain-of-function mutant LRRK2 (G2019S) inhibited the uncoating of AP2M1 from CCVs at later stages and prevented new cycles of CCV formation. Thus, the abundance and activity of LRRK2 must be calibrated to ensure proper endocytosis. Dysregulated phosphorylation of AP2M1 from the brain but not thyroid tissues of LRRK2 knockout and G2019S-knockin mice suggests a tissue-specific regulatory mechanism of endocytosis. Furthermore, we found that LRRK2-dependent phosphorylation of AP2M1 mediated dopaminergic neurodegeneration in a Drosophila model of PD. Together, our findings provide a mechanistic link between LRRK2, AP2, and endocytosis in the pathogenesis of PD.

Vapor-Phase Plotting of Organosilane Chemical Gradients.

Vapor-phase plotting of organosilane-based self-assembled monolayer (SAM) gradients is demonstrated for the first time. Patterned SAMs are formed by delivering gas-phase organotrichlorosilane precursors to a reactive silica surface using a heated glass capillary. The capillary is attached via a short flexible tube to a reservoir containing the precursor dissolved in toluene. The proximal end of the capillary is positioned at an experimentally optimized distance of 30 µm above the substrate during film deposition. The capillary is mounted to a stepper-motor-driven X, Y plotter for raster scanning above the surface. Two different organotrichlorosilane precursors are employed in this initial demonstration: n-octyltrichlorosilane and 3-cyanopropyltrichlorosilane. The dependence of SAM deposition on ambient relative humidity, capillary-substrate separation, raster-scanning speed, and solvent viscosity and volatility is explored and optimum deposition conditions are identified. The optimized procedures are used to plot uniformly modified square "pads" and gradients of the silanes. Film formation is verified and the gradient profiles are obtained by sessile drop water contact angle measurements, spectroscopic ellipsometry measurements of film thickness, and X-ray photoelectron spectroscopy mapping. The resolution of the plotting process is currently in the millimeter range and depends on capillary diameter and distance from the substrate surface. Vapor-phase plotting affords a unique direct-write method for producing patterned and chemically graded SAMS that may find applications in microfluidic devices, planar chromatography, and optical and electronic devices.