Interaction between the astrocytic glutamate transporter, EAAT2, and protein phosphatase 2A

The concentration of extracellular glutamate is maintained below neurotoxic levels by the family of sodium-dependent high affinity glutamate transporters. The astrocytic glutamate transporter, EAAT2, is responsible for 90% of all glutamate clearance from the synapse in the mammalian CNS. Elucidating the various mechanisms that can play a role in regulating glutamate transporter activity is critical for our understanding of how the transporters maintain glutamate homeostasis. The primary objective of this study was to identify putative EAAT2-interacting proteins, and to characterize the effect of this interaction on glutamate transporter function under control and neurodegenerative conditions. Utilizing a Bac-2 Hybrid screen, we identified the serine/threonine protein phosphatase, PP2A, as a putative EAAT2-interacting protein. We confirmed this interaction both in vivo, and in vitro, and determined that the interaction between EAAT2 and PP2A was specific to the reentrant loop moiety of the transporter. Inhibition of PP2A by okadaic acid, resulted in subcellular redistribution of both PP2A and EAAT2 protein. Specifically, PP2A redistributed from the cytoplasm to the nucleus, while EAAT2 levels increased in the cytoplasm. This change in subcellular localization was accompanied by increased glutamate transport activity in both cortical and spinal cord astrocyte and neuronal co-cultures, and increased cell surface expression of EAAT2, particularly in cortical astrocytes. We also examined the effects of this novel protein interaction under neurodegenerative conditions using the SOD1G93A mouse model of ALS. Within the spinal cord, PP2A inhibition caused a similar redistributrion of PP2A to the perinuclear area and significant increased glutamate transport activity. These effects were not seen in cortical cultures, suggesting that the interaction between PP2A and EAAT2 may play a more significant role in the spinal cord. Further research is needed to fully understand the possible functions of this interaction in neurodegenerative states, and may ultimately lead to the development of novel therapeutic strategies for the treatment of ALS.