A LRRK2-Dependent EndophilinA Phosphoswitch Is Critical for Macroautophagy at Presynaptic Terminals

Abstract

Manuscript 2Synapses are often far from the soma and independently cope with proteopathic stress induced by intense neuronal activity. However, how presynaptic compartments turnover proteins is poorly understood. We show that the synapse-enriched protein EndophilinA, thus far studied for its role in endocytosis, induces macroautophagy at presynaptic terminals. We find that EndophilinA executes this unexpected function at least partly independent of its role in synaptic vesicle endocytosis. EndophilinA-induced macroautophagy is activated when the kinase LRRK2 phosphorylates the EndophilinA-BAR domain and is blocked in animals where EndophilinA cannot be phosphorylated. EndophilinA INTRODUCTIONNeurons can be metabolically very active, firing at rates of more than 100 Hz.Synaptic proteins and organelles are used and re-used multiple times and accumulate damage as a result of this stress. Furthermore, synapses are often located far away from the cell body and must therefore in part operate independently. This raises the question how synapses maintain protein quality. Given that neurodegeneration is thought to start with subtle synaptic defects before evolving into blunt neuronal death (Burke and O'Malley, 2013;, the mechanisms of synaptic protein homeostasis are likely relevant for the understanding of neurodegenerative disease.Macroautophagy is well-placed to mediate protein turnover, but how the needs of synapses are served by this process has not been well-studied. Cellular signals like stress and amino acid deprivation induce macroautophagy, where cytoplasm is engulfed by double membrane structures before fusion with degradative lysosomes (Mizushima et al., 2011).Autophagosomes have been visualized using fluorescent markers in yeast, Drosophila and mammalian cells and are often observed as Atg8/LC3 positive puncta (Kabeya et al., 2000;Scott et al., 2004). At the stage of initiation, Atg9-positive vesicles fuse into elongated preautophagosomal structures with growing edges (He et al., 2006). These edges are highly curved and harbor lipid packing defects. These edges serve as protein docking sites, attracting specific autophagic factors such as Atg3, Atg14/Barkor and Atg1 that insert into such zones, recognizing specific lipids (phosphatidylinositol-3-phosphate (PI(3)P)) and lipid packing defects (Fan et al., 2011;Nath et al., 2014;Ragusa et al., 2012). The recruitment of these factors then promotes the further steps of autophagosome formation; in particular the E2-like protein Atg3 itself recruits the autophagic marker LC3/Atg8 (Nath et al., 2014). However, how these highly curved edges are formed and maintained is very poorly understood.While autophagy has been mostly analyzed in the soma of cultured cells and yeast, autophagic markers have also been observed away from the soma at neuronal synapses 4 (Hernandez et al., 2012;Maday and Holzbaur, 2014;Williamson et al., 2010) and these markers were shown to be transported along axons (Maday and Holzbaur, 2014). However, how autophagosomes are formed at synapses an...

Endophilin-A Deficiency Induces the Foxo3a-Fbxo32 Network in the Brain and Causes Dysregulation of Autophagy and the Ubiquitin-Proteasome System

“…Thus, endophilin and FBXO32 membrane-tubulating activity may contribute to the formation/maturation of autophagosomes. This model is in concordance with the reported role of endophilin-A as necessary and sufficient for macroautophagy at presynaptic terminals of fruit flies (Soukup et al., 2016). …”

Parkinson Sac Domain Mutation in Synaptojanin 1 Impairs Clathrin Uncoating at Synapses and Triggers Dystrophic Changes in Dopaminergic Axons

“…We have now found that a similar up-regulation of parkin occurs in SJ1 RQ -KI brains, strongly linking the function of parkin to that of both endophilin and synaptojanin. Other links between PD proteins and the endocytic pathway include the following: 1) LRRK2 has been implicated in endocytic and endosomal function (Beilina et al, 2014; MacLeod et al, 2013; Schreij et al, 2015; Steger et al, 2016), and most interestingly in the context of the present study, it regulates endophilin and SJ1 by phosphorylation (Arranz et al, 2015; Islam et al, 2016; Matta et al, 2012; Soukup et al, 2016). 2) Loss of α-synuclein leads to increased level of endophilin (Westphal and Chandra, 2013).…”

Malfunctions in synaptic membrane trafficking in early pathology of Parkinson’s disease: New molecular clues

“…It is altered in the cortex of PD patients, and it interacts with two hallmark PD proteins, the E3 ubiquitin ligase parkin and the leucine-rich repeat kinase LRRK2, the most commonly disrupted gene in familial PD (Murdoch et al, 2016;Soukup et al, 2016;Soukup and Verstreken, 2017). Unbiased proteomic screening of brain proteins in mice lacking all three synuclein genes revealed a prominent increase in endophilin 1 levels (Burre et al, 2013).…”

“…Several recent studies linked endophilin and its endocytic binding partner synaptojanin to the maturation of autophagosomes in the synapse (Murdoch et al, 2016;Soukup et al, 2016;Soukup and Verstreken, 2017), which expanded its role far beyond the SV recycling and synaptic compartment, and allowed linking it to pathological neurodegenerative conditions, including PD, in accordance with the complex phenotype of the endophilin TKO (Fig. 1).…”

“…1). Endophilin 1 localizes on autophagosomal membranes and is critical for the maturation of synaptic autophagosomes (Murdoch et al, 2016;Soukup et al, 2016).…”