Error bar represents SEM. In contrast to the elevation of Parkin, the levels of two other enzymes of ubiquitin metabolism, Nedd4 (another E3 ubiquitin ligase) and UchL1 (a de-ubiquitinating enzyme expressed in nerve system also implicated in PD), were present at normal concentrations in endophilin TKO brains (data not shown). The function of endophilin is closely interconnected with the function of synaptojanin 1, as (1) these two proteins bind to each other; (2) the presence of endophilin helps target and stabilize synaptojanin 1; and (3) the absence of endophilin or of synaptojanin produces striking similar phenotypes in worms, flies, and mice (Schuske et al., 2003;Verstreken et FANCG al., 2003;Hayashi et al., 2008;Milosevic et al., 2011). with the identification of a PD-linked synaptojanin 1 mutation, in providing evidence for a link between PD and endocytosis Serlopitant genes. Keywords:Parkinson, Parkinsonism, dynamin, clathrin, auxilin == Introduction == Synaptic transmission relies on the release of neurotransmitters Serlopitant via the fusion of synaptic vesicles with plasma membrane. Rapid endocytosis and recycling of synaptic vesicles allow synapses to function even during high-frequency stimulation. Endophilin, the collective names of endophilin 1, 2, and 3 in mammals, has an important role in this process as an adaptor that recruits other endocytic factors to endocytic sites. Its N-terminal BAR domain binds high-curvature membranes (the neck of endocytic pits), while the two major binding partners of its SH3 domain are dynamin, a GTPase implicated in the fission of the bud neck, and synaptojanin 1, the phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] phosphatase required for rapid clathrin uncoating after fission (de Heuvel et al., 1997;Ringstad et al., 1997;Cremona et al., 1999). The interaction of endophilin with synaptojanin is particularly important as the absence of endophilin or of synaptojanin produces strikingly similar phenotypes in worms, flies, and mice (Schuske et al., 2003;Verstreken et al., 2003;Hayashi et al., 2008;Milosevic et al., 2011). In mice, the absence of the three endophilins or of synaptojanin 1 results in perinatal lethality Serlopitant and in a dramatic accumulation of clathrin-coated vesicles at synapses (Hayashi et al., 2008;Milosevic et al., 2011). Partial loss of endophilin [endophilin 1 and 2 double KO (DKO) mice] is compatible with early postnatal life but results in neurodegeneration and death within 3 weeks (Milosevic et al., 2011). Parkin is an E3 ubiquitin Serlopitant ligase whose mutations lead to autosomal recessive juvenile-onset PD (Shimura et al., 2000). Parkin comprises an N-terminal ubiquitin-like domain (UBL), a central RING0 domain, and a C-terminal RING1-IBR-RING2 domain. Parkin can catalyze both K48-linked poly-ubiquitination for proteasome-mediated degradation and K63-linked poly-ubiquitination and mono-ubiquitination for the regulation of proteinprotein interactions (Dawson and Dawson, 2010). Several synaptic proteins were identified as Parkin’s substrates and/or interactors. These include the synaptic vesicle-associated proteins CDCrel-1 and synaptotagmin XI, which are degraded through Parkin-mediated poly-ubiquitination (Zhang et al., 2000;Huynh et al., 2003), and CASK, PICK1, and Eps15, which are modified through Parkin-dependent mono-ubiquitination (Fallon et al., 2002,2006;Joch et al., 2007). They also include endophilin, which interacts with the UBL domain of Parkin via its SH3 domain and is ubiquitinated by Parkin. This interaction is regulated by phosphorylation, suggesting that it may be part of a regulatory mechanism (Trempe et al., 2009). These findings are of significant interest considering that biochemical and genetic interactions have been observed between endophilin and LRRK2, another protein implicated in PD (Matta et al., 2012), and that a mutation in the preferred endophilin partner, synaptojanin 1, was observed in PD patients (Krebs et al., 2013;Quadri et al., 2013;Olgiati et al., 2014). Here we show that Parkin’s protein level is specifically upregulated in brain tissue and cells of mice lacking endophilin. Furthermore, we show that not only endophilin itself, but also its major binding partners, can be mono-ubiquitinated by Parkin. == Materials and Methods == == == == == == Animals and tissues. == Endophilin 1 and 2 DKO and endophilin triple KO (TKO) mice were described previously (Milosevic et al., 2011). Parkin KO mice were obtained from The Jackson Laboratory (006582, B6.129S4-Park2
