PRRT2 Regulates The Molecular Mechanism Of The Release Of Amino Acid Neurotransmitter Vesicles Through The SNARE Complex

Paroxysmal kinesigenic choreoathetosis(PKC,OMIM 128200)is a relatively rare neurological movement disorder with the traits of recurrent,brief attacks of abnormal involuntary movements induced by sudden voluntary movements.The seizure onset usually occurs at the childhood or the preadolescence.The episodes usually present with unilateral or bilateral involuntary movements,such as chorea,athetosis or dystonia.The disease was firstly reported by Kertesz in 1967.Tomita defined the disease locus within the region of 16p11.2-q12.1.Many researchers including our group confirmed that the proline-rich transmembrane protein 2(PRRT2)was associated with various neurological diseases,including PKC,benign familial infantile epilepsy(BFISs),infantile convulsions and choreoathetosis(ICCA)and so on,which were all termed as "PRRT2-related disorders".Studies on mice revealed that Prrt2 is specifically expressed in nervous tissues with especially high level in brain and spinal cord.PRRT2 with a proline-rich region(PRD)is a protein of 340 amino acids existing two transmembrane helices.PRRT2 interacts with SNAP25,synaptotagmin as well as GRIA1(a subunit of the ionotropic glutamate receptors AMPA).PRRT2 could affect the function of synapses by regulating the assembly of SNARE complex.Specific knockout of Prrt2 in the cerebellum of mice leads to abnormal synaptic transmission.All conclusions above hint that PRRT2 may take part in the release of neurotransmitters and affect synaptic transmission.However,the molecular mechanisms of how the PRRT2 mutant leads to the abnormal synaptic transmission and triggers PKC are still unclear.This study aimed to research the molecular mechanisms of PRRT2 regulating the release of the amino acid neurotransmitter vesicles through SNARE complex.1.A truncated mutant rat model knocked out 460bp in the second exon(80th-539th bp)of Prrt2 gene by CRISPR/Cas9-mediated gene editing technology was generated.The identification results of Prrt2 gene and PRRT2 protein level in the rat model indicated that Prrt2 gene was truncated successfully in the rat model.2.Immunofluorescence(IF)staining results confirmed that PRRT2 was selectively expressed in neuron in rat.The results of IF combined with sucrose density gradient ultracentrifugation indicated that PRRT2 expressed on both pre-and post-synaptic membrane.3.Compared to Prrt2+/+ rats,the protein levels of the SNARE elements(SNAP25,VAMP2,STX1A)and SYT1 all rose significantly in the cortex Ml region of Prrt2-/-rats(SYT1,P=0.0394;SNAP25,P=0.0016;STX1A,P=0.0007;VAMP2,P=0.0019).The results of co-immunoprecipitation(co-IP)indicated that PRRT2 interacted with SNAP25,VAMP2 and STX1A.Compared to Prrt2+/+ rats,the interaction between SNAP25 and VAMP2 was significantly high in the cortex M1 region of Prrt2-/-rats(SNAP25,P=0.0011;VAMP2,P=0.0039).This result indicated that the assembly of SNARE complex increased.4.Microdialysis and HPLC experiments results confirmed that in the cortex M1 region of Prrt2-/-rats,the levels of the amino acid neurotransmitters(ASP,P=0.0362;GLU,P=0.0474;GLY,P=0.0224;GABA,P=0.0331)in extracellular fluid ascended.5.The results of co-IP showed that PRRT2 interacted with GRIA1.Compared to Prrt2+/+ rats,the protein level of glutamate receptors GRIA1 in the cortex M1 region of Prrt2-/-rats was significantly high(PCX=0.0099;PHC=0.0004),while it was as opposite as the GABA receptors GABRA1(P=0.0145)and GABRA5(P=0.0006).6.Western Blot results showed that compared to Prrt2+/+ rats,the protein level of NR2B in the cortex Ml region and hippocampus was high(PCX=0.0309;PHC=0.0393),while the protein level of NR2A in the cortex M1 region was low in the Prrt2-/-rats(P=0.0228).Y-maze test result showed there was spatial learning and memory impairment in Prrt2-/-rats(P=0.0053).All the results mentioned above indicate that PRRT2 could regulate the vesicle release of the amino acid neurotransmitters through SNARE and affect the protein level of glutamate receptors and the GABA receptors.PRRT2 mutant leading to the imbalance between excitatory and inhibitory neurons is associated with PKC phenotype.