LRRK2 controls an EndoA phosphorylation cycle in synaptic endocytosis

During the last decade several gene mutations have been identifiedwhich cause inherited forms of Parkinson's disease. While Parkinsons disease is mostly a sporadic disease, understanding the function of these genes and how mutations affect their activity may contribute to the understanding of the pathogenesis of the disease and hopefully may lead to the development of therapies that can slowdown the progression of the disease. In this thesis we study LRRK2, the most common mutated gene in Parkinson's disease. Using the fruit fly as model system, we show that the loss of LRRK2 expression leads to a decrease in endocytosis of synaptic vesicles highlighting the importance of LRRK2 in assuring neuronal communication. The fruit fly is a very attractive model to study gene functions, especially because crosses between fruit flies harboring different genetic mutations can be quickly generated. By crossing flies with a mutated LRRK2 gene and flies with mutations in other genes implicated in synaptic vesicle endocytosis, we find that a decreased level of endophilin A, a key player in Clathrin mediated endocytosis (CME) rescue the phenotype caused by loss of LRRK2. This suggests that LRRK2 and Endophilin A cooperate to regulate synaptic function. Furthermore, we show that LRRK2 regulates Endophilin A function by phosphorylating residue Serine 75 in its (BAR) domain. Due to its unique (BAR) domain structure, endophilin can recognize, bind and tubulate membranes in vitro. In neurons, endophilin cooperates with other proteinsto maintain neurotransmission. By using different endophilin phospho-mimetic Serine 75 mutants, we show that LRRK2 phosphorylation interferes with the ability of Endophilin A to interact with the plasma membrane which is very important to assure the renewal of synaptic vesicle during neurotransmission. Flies containing the phospho-mimetic endophilin Serine 75 transgene show indeed defects in endocytosis and neurotransmission similar to the ones containing the LRRK2 G2019S mutation known to have an increased kinase activity. The reduced phosphorylated Serine 75 levels in LRRK2-deficient flies, but also the excessive presence of phosphorylated Serine 75 in LRRK2-G2019S lead both to a defect in endocytosis and synaptic transmission, indicating that too much or too little Serine 75 phosphorylation in endophilin A leads to a defect in endocytosis and neurotransmission. In support of this model, we show that inhibition of the kinase activity of LRRK2-G2019S LRRK2 in flies using LRRK2-IN1, a selective inhibitor of LRRK2 rescues the synaptic deficit. This effect is dose dependent and when excess LRRK2-IN1 is administered also inhibition of vesicle recycling is observed, as anticipated by the model. In conclusion, we show that a correct balance of phosphorylated and non-phosphorylated Endophilinat Serine 75 is crucial for proper regulation of synaptic vesicle recycling in neurons, which is regulated by the kinase activity of LRRK2. We speculate that imbalance of this phosphorylation event may result in Parkinson's disease. It is clear that the development of LRRK2 inhibitors as a therapy for Parkinsons disease might be complicated because too little or too much of drug might disturb this balance in a pathological way.status: publishe