PRRT2 Mutants Lead To Glutamate Release And GRIA1 Membrane Surface Distribution Abnormalities

Paroxysmal kinesigenic choreoathetosis (PKC, OMIM 128200) is the most commonly described type of paroxysmal dyskinesias disorder also known as paroxysmal kinesigenic dyskinesia (PKD) and was first described in 1967. The major clinical phenotype of PKC is sudden attacks of involuntary movement, such as dystonic posturing, chorea or athetosis which is often induced by sudden movement from the rest, or change in velocity of movement. In 2011, our team and other researchers indentified the heterozygous mutations on the proline-rich transmembrane (TM) protein 2 (PRRT2) gene at 16p11.2 are responsible for PKC. PRRT2 (Q7Z6L0) is predicted to consiste of an N-terminal extracellular domain containing a proline-rich domain and an N-glycosylation site, two TM domains in its C-terminal sequence, which was extremely conserved across species. In the study on mice, researchers found that PRRT2 mainly expressed in nervous system with high levels present in the cerebral cortex, hippocampus and cerebellum.Although little is known about the function of PRRT2, Lee et al. confirmed the interaction between SNAP25 and PRRT2 by coimmunoprecipitation experiments, they also found that there is no expression of the mutant forms (p.R217Pfs*8) of PRRT2 which is the most frequent mutation found in patients. SNAP25 is a t-SNARE presynaptic protein implicated in the formation of the neuronal exocytotic fusion apparatus and plays important role for the regulation of excitatory animo acids (EAAs) neurotransmitter relaease. The dysfunction of EAAs transmission especially glutamate plays roles in many nervous diseases, including epilepsy, atuism and migraine. Another important clue is that a high-resolution proteomics study indentified 21 novel protein constituents which contains of PRRT2 of native α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor complexes in 2012. Moreover, PRRT2 is preferentially associated with the inner submit GRIA1.To study the function of PRRT2 protein, we first measured three kinds of EAAs levels in the plasma of 7 PKC patients and 12 mached healthy controls via HPLC and found that both the glutamate and asptartate levels in the plasma of PKC patients is higher comparing with the mached health controls. Subsequently, we performed a loss-of-function experiments using the shRNA lentivirus targeting Prrt2 (shRNA-Prrt2) infecting the primary cultured mouse cotex neurons. We measured the three kinds of EAAs levels in the culture medium of neurons after infection and found that the glutamate level is increased in the shRNA-Prrt2 group comparing with pLL3.7 control group. These results indicated that PRRT2 involved in the released of glutamate and probably as an inhibitory factor. Moreover, our multi-immunostaining assry showed that Prrt2 located at the glutamatergic terminals in accordance with its function.For the next, we constructed the over expression vectors of PRRT2 WT, p.R217Pfs*8, and p.A287T where the later one was considered as the missense mutation found by our lab to further examined the interactions between SANP25. The coimmunoprecipitation experiments results revealed that the interaction between SNAP25 and PRRT2 is weaked by the missense mutations, suggesting that this might be the way for PRRT2 to inhibit the released of glutamate.To futher exploring the partners interacting with PRRT2, we proved the interaction between GRIA1 and PRRT2 for the first time both in vivo and vitro by coimmunoprecipitation experiments. We also found that the interaction between GRIA1 and mutant PRRT2 decreased comparing with the wild type, but the mutant PRRT2 increased the membrane distribution of GRIA1.In summary, we have identified the causative gene of PKC, but the mechanism for mutant PRRT2 leading to PKC is still poorly understood. In the present study, we found higher glutamate levels in the plasma of PKC patients and in the culture medium of neurons when Prrt2 expression was knocked down. We further found that the interaction of PRRT2 with SNAP25 was disturbed by the missense mutation of PRRT2, which suggesting the way that PRRT2 mediates its function. At last, we proved the interaction between PRRT2 and GRIA1 for the first time. Moreover, the membrane distribution of GRIA1 was increased when interacted with the mutant form of PRRT2. Our results showed that muatant form of PRRT2 leads to dysfunction of glutamate signaling...