Conversely, adding even higher doses of LRRK2-IN1 should block both endogenous LRRK and LRRK2G2019S and again impede endocytosis. As shown in Figure 8B, we find that the defect in synaptic vesicle endocytosis upon application of LRRK2-IN1 to LRRK2G2019S-expressing animals is rescued. Interestingly, further increasing the LRRK2-IN1 concentration applied to LRRK2G2019S-expressing animals again results in defects to internalize FM1-43 ( Figure 8B). Taken together, the data indicate that both increased and decreased LRRK-dependent
EndoA phosphorylation impedes synaptic vesicle endocytosis ( Figures 8C and 8D). In this work, we show that EndoA is phosphorylated by LRRK2 and that this posttranslational modification modulates EndoA-dependent FRAX597 chemical structure membrane deformation in vitro and endocytosis of synaptic membrane in vivo. Endocytosis is a dynamic process that depends on numerous transient protein-protein and
protein-lipid interactions (Schmid et al., 2006; Tonikian et al., 2009). Our data indicate that reduced S75 phosphorylation in EndoA, although still supporting membrane tubulation find more in vitro, acts negatively on synaptic recycling in vivo, because the protein fails to efficiently leave the membrane (Figure 8D). In line with this, a constitutive membrane-bound EndoA (Unc-57) also does not fully rescue unc-57 C. elegans mutants ( Bai et al., 2010). In addition, overexpression of EndoA in Lrrk mutants exacerbates synaptic vesicle endocytosis, while heterozygous loss of endoA rescues Lrrk mutants. The data suggest that excessive dephosphorylated EndoA in Lrrk flies is “locked” such that constitutive membrane binding of the protein impedes the transient and vibrant interactions needed to create new vesicles and/or to Tolmetin deliver the protein to endocytic zones ( Bai et al., 2010). EndoA is also involved in recruiting Synaptojanin (Synj) to nascent synaptic vesicles. Synj is a phosphoinositide phosphatase involved in uncoating of
proteins from newly formed vesicles, and endoA null mutant synapses display reduced levels of Synj and an increased number of densely coated synaptic vesicles ( Milosevic et al., 2011; Schuske et al., 2003; Verstreken et al., 2002). Given that it is difficult to discern coated from uncoated vesicles in our TEM preparations of the EndoA S75 phosphomutants, we did not determine whether the amount of densely coated vesicles is altered. However, we observe reduced levels of the uncoating factor Synaptojanin in Lrrk animals (unpublished data), suggesting that in the Lrrk flies late stages of endocytosis may also be blocked. Further work is needed to determine precisely how different aspects of EndoA function are affected by LRRK.
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