LRRK2 phosphorylates Snapin and inhibits interaction of Snapin with SNAP-25

Abstract

Leucine-rich repeat kinase 2 (LRRK2) is a gene that, upon mutation, causes autosomal-dominant familial Parkinson's disease (PD). Yeast two-hybrid screening revealed that Snapin, a SNAP-25 (synaptosomal-associated protein-25) interacting protein, interacts with LRRK2. An in vitro kinase assay exhibited that Snapin is phosphorylated by LRRK2. A glutathione-S-transferase (GST) pull-down assay showed that LRRK2 may interact with Snapin via its Ras-of-complex (ROC) and N-terminal domains, with no significant difference on interaction of Snapin with LRRK2 wild type (WT) or its pathogenic mutants. Further analysis by mutation study revealed that Threonine 117 of Snapin is one of the sites phosphorylated by LRRK2. Furthermore, a Snapin T117D phosphomimetic mutant decreased its interaction with SNAP-25 in the GST pull-down assay. SNAP-25 is a component of the SNARE (Soluble NSF Attachment protein REceptor) complex and is critical for the exocytosis of synaptic vesicles. Incubation of rat brain lysate with recombinant Snapin T117D, but not WT, protein caused decreased interaction of synaptotagmin with the SNARE complex based on a co-immunoprecipitation assay. We further found that LRRK2-dependent phosphorylation of Snapin in the hippocampal neurons resulted in a decrease in the number of readily releasable vesicles and the extent of exocytotic release. Combined, these data suggest that LRRK2 may regulate neurotransmitter release via control of Snapin function by inhibitory phosphorylation.

LRRK2 functions in synaptic vesicle endocytosis through a kinase-dependent mechanism

“…Recently, it has been described that LRRK2 could also modulate synaptic vesicle exocytosis, either by regulating presynaptic vesicle release or by interacting with snapin (Piccoli et al, 2011;Yun et al, 2013). Yun and colleagues claimed that LRRK2-dependent phosphorylation of snapin decreased the extent of exocytotic release in hippocampal neurons as measured by using vGlut-phluorin assays.…”

“…Substrates are involved in different processes, such as neuronal survival, cytoskeletal rearrangement, autophagy and apoptosis (Berwick and Harvey, 2011;Gillardon, 2009;Plowey and Chu, 2011;Martin et al, 2014). LRRK2 has also been shown to regulate neurotransmission, by interacting with synaptic proteins that modulate clathrin-mediated endocytosis of synaptic vesicles, such as Rab5b, snapin and N-ethylmaleimide-sensitive factor (NSF) (Piccoli et al, 2011;Shin et al, 2008;Yun et al, 2013;Piccoli et al, 2014). Moreover, we revealed an essential role for LRRK in synaptic vesicle endocytosis at Drosophila melanogaster neuromuscular junctions through phosphorylation of endophilin A (EndoA) (Matta et al, 2012).…”

Cellular processes associated with LRRK2 function and dysfunction

“…LRRK2 has been proposed to participate in the control of synaptic vesicle exocytosis by phosphorylating Snapin, and thus regulating soluble NSF attachment protein receptor (SNARE) complex functionality and late endosomal transport 125. Alterations in the amount of ready releasable vesicles have been described in cell models overexpressing G2019S‐ LRRK2 126.…”

Flies with Parkinson's disease

“…These findings in fruit flies, but also the work in many other systems strongly implicate a group of PD-relevant proteins rather directly in (synaptic) vesicle trafficking steps (Braschi et al, 2010;Cirnaru et al, 2014;Gitler et al, 2008;McLean et al, 2000;Piccoli et al, 2011;Soper et al, 2011;Vargas et al, 2014;Yun et al, 2013). This work then opens the question as to how defects in vesicle trafficking can be connected to PD?…”