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  • Addiction, Drugs
  • Information from Lay-Language Summaries is Embargoed Until the Conclusion of the Scientific Presentation

    423—Neurotransmitter Release: Vesicle Recycling and Biogenesis

    Monday, November 11, 2013, 1:00 pm - 5:00 pm

    423.12: Genetic interaction between endophilin and Parkin

    Location: Halls B-H

    *M. CAO1,2,3, I. MILOSEVIC1,2,3, S. GIOVEDI1,2,3, P. DE CAMILLI1,2,3;
    1Yale Univ., New Haven, CT; 2Program in Cell. Neuroscience, Neurodegeneration and Repair, New Haven, CT; 3Howard Hughes Med. Inst., New Haven, CT

    Abstract Body: Release of neurotransmitter at synapses is mediated by the fusion of synaptic vesicles (SVs) with plasma membrane. Rapid endocytosis and recycling of SV membranes allows synapses to function reliably even during high frequency stimulation. A protein that plays an important role in this process is the BAR domain containing protein endophilin (the collective name for endophilin 1, 2 and 3), an endocytic adaptor with curvature generating and curvature sensing properties that helps recruit the GTPase dynamin and the polyphosphoinositide phosphatase synaptojanin to the collar of endocytic pits. While the deletion of all three endophilins genes leads to perinatal lethality, mice lacking selectively the endophilin 1 and 2 genes survive at birth but die within three weeks due to early neurodegeneration. Parkin, an E3 ligase that functions in the ubiquitin/proteasome pathway, was shown to interact directly with endophilin. As mutations of Parkin in humans lead to autosomal recessive juvenile-onset Parkinson’s disease (PD), a neurodegenerative disease, we explored a potential genetic interaction between the two proteins. We have found that Parkin’s levels are specifically up-regulated in the brain of both newborn endophilin1/2/3 triple and endophilin 1/2 double knockout (KO) mice. Furthermore, endophilin 1/2 double KO mice that also are KO for the Parkin gene display a more severe neurological phenotypes and shorter lifespan than endophilin 1/2 double KO mice. Studies of transfected HEK293T cells show that Parkin not only ubiquitinates (primarily monoubiquitinates) endophilin, but also dynamin and synaptojanin, while PD-linked Parkin mutants fail to do so. These results complement recent findings demonstrating a genetic link between endophilin and another PD protein LRRK2 and provide new evidence for a link between neurodegeneration and endocytosis genes.

    Lay Language Summary: Our research builds on two previous findings: 1) the discovery of an interaction between the endocytic protein endophilin and the E3 ubiquitin ligase Parkin, aParkinson’s disease-linked protein; 2) the identification of an early neurodegenerative phenotype in mice with lower levels of expression of endophilin due to ablation of two of the three endophilin genes. We now report that Parkin levels are specifically up-regulated in endophilin knockout mice brains, and the newly-generated Parkin/endophilin1/2 triple knockout mice display more severe neurological phenotypes and shorter lifespan than endophilin 1/2 double knockout mice. We also identify two endophilin interacting proteins dynamin and synaptojanin 1 as the new substrates of Parkin. The potential impact of these results is strikingly enhanced by the recent identification of a synaptojanin 1 mutation in human patients with early onset Parkinsonism.
    Communication between neurons in the brain is greatly impaired in patients with Parkinson’s disease, and relies on the correct balance of neurotransmitters, such as dopamine, in the space between neurons. Release of neurotransmitter at synapses is mediated by the fusion of synaptic vesicles with plasma membrane. Rapid internalization (endocytosis) and recycling of synaptic vesicle membranes is crucial to regulate the extracellular availability of neurotransmitters that act on target neurons, and to prime the cell with a ready store of neurotransmitters, enabling quick response to further rapid stimulation. Post-synaptically, membrane traffic, including endocytosis, is required to control the number of neurotransmitter receptors exposed at the cell surface and thus to fine tune the response to neurotransmitter signals. However, how defects in endocytosis may contribute to Parkinson’s disease is not well understood. A key factor implicated in the endocytosis of synaptic vesicles at synapses is endophilin. Endophilin localizes at the neck of endocytic buds and helps recruit other endocytic factors such as the GTPase dynamin and the PI(4,5)P2 phosphatase synaptojanin 1, which are needed for vesicle fission and vesicle uncoating, respectively. Recently, endophilin was shown to bind to the E3 ubiquitin ligase Parkin. Interestingly, mutations in Parkin lead to autosomal-recessive juvenile-onset Parkinson’s disease. Based on these previous finding, we suggest that endophilin helps recruit Parkin to endocytic sites of synapses, where Parkin-mediated ubiquitination may regulate protein-protein interactions and control membrane traffic.
    To test this hypothesis, we first measured the protein level of Parkin in endophilin knockout mice brain. Surprisingly, Parkin level was found specifically increased in endophilin1/2/3 triple and endophilin1/2 double knockout mice, but not in other
    endocytic factor knockout or neurodegenerative model mice. Previously, we reported that endophilin 1/2 double knockout mice die within 3 weeks due to early neurodegeneration. To test whether Parkin is involved in this neurodegeneration process, we generated Parkin/endophilin1/2 triple knockout mice. Interestingly, these triple knockout mice show more severe neurological phenotypes, delayed nerve system development and they usually die within 24 hours after birth, compared with endophilin1/2 double knockout mice. These results further confirmed the genetic interaction between Parkin and endophilin. To explore more about the mechanism of this interaction, we also checked the capability of Parkin to ubiquitinate different endocytic proteins by co-expressing them with ubiquitin in HEK293T cells. Not only endophilin itself, but also its strong binding partners dynamin and synaptojanin 1, were found to be mono-ubiquitinated by wild-type but not PD-linked mutants of Parkin, suggesting Parkin could regulate endophilin complex formation.
    Complemented with recent findings showing a genetic link between endophilin and another Parkinson’s disease protein Lrrk2, and the identification of mutation in synaptojanin 1 gene associated with autosomal-recessive juvenile-onset Parkinson’s disease, our study shed light on the overall importance of membrane trafficking, including endocytosis, in Parkinson’s disease and provide a completely novel perspective on pathogenetic mechanisms of Parkinson’s disease.