The Role Of Lrrk2 In The Modulation Of The Glutamate Transporter Eaat2 In Parkinson'S Disease

Parkinson's disease (PD) is a common chronic and progressive neurodegenerative disorder characterized by a complex physiopathology. Although several known causes lead to the onset of the disease, a central role has been attributed to excitotoxicity and subsequent cellular damages. Glutamate-mediated excitatory synaptic signalling is mainly directed by excitatory amino acid transporter 2 (EAAT2), localized on astrocyte plasma membrane and involved in the glutamate clearance. Alterations in the function or in the expression of EAAT2 lead to extracellular glutamate accumulation, playing an important role in the PD's physiopathology. One of the most common mutation in PD patients is G2019S substitution in Leucin Rich Repeat Kinase 2 (LRRK2). Recently, an impaired cortico-striatal glutamatergic transmission in mice carrying G2019S mutation has been reported. The role of LRRK2 wild-type and mutated in regulating the EAAT2 function and cellular localization have been investigated by heterologous expression in Xenopus laevis oocytes. The Two Electrodes Voltage Clamp (TEVC) and immunochemistry were applied to collect data about the activity and the protein expression. The results demonstrate that LRRK2 acts on EAAT2 significantly increasing the inward glutamate transport current when the wild-type protein is present. Conversely, testing the transporter with the pathogenic LRRK2 G2019S kinase elicited transport currents with amplitude similar to that of EAAT2 expressed alone. The LRRK2 kinase inhibitor MLi-2 can revert the phenotype of the mutated protein G2012S, showing in this condition a transport current amplitude similar to that recorded in the presence of the LRRK2 wild-type. The apparent substrate affinity of EAAT2 for glutamate was higher when co-expressed with G2012S than with wild-type LRRK2, slowing down the glutamate transport cycle. The immunochemistry study on oocytes heterologously expressing EAAT2 with LRRK2 wild-type and G2012S suggested that the amount of transporter on the plasma membrane is reduced in the presence of the mutated protein, while MLi-2 treatment restored the transporter localization. The electrophysiological studies and immunochemistry results highlight that the reduced membrane localization of transporter and the higher affinity can lead to an impaired clearance of glutamate, resulting in the well-known pathological effects. Our data confirmed the functional interaction between EAAT2 and LRRK2 and support the idea that LRRK2 G2019S could be responsible of chronic glutamate accumulation and neurodegeneration in PD.