The Mechanism Of Plasticity Related Gene-1 Uptake With Extracellular Lysophosphatidic Acid

Recently, a new class of membrane protein named Plasticity Related Genes(PRGs), which are specific expressed in vertebrate brain have been identified by Nitsch group. These protein exhibit highly homologic and similar structure to the lipid phosphate phosphatase superfamily, which can generate biologic lipid phosphate. Plasticity Related Gene- 1(PRG1), the first named PRGs family, is the first expressed at E19 in the hippocampus, which are three extracellularly located and catalytic domains used to dephosphorylate lysophosphatidic acid(LPA). Its C-terminal and N-terminal are intracellular. Functional analyses and sequence alignments of PRG1/LPPR4 with regard to its ecto-enzymatic activity revealed increased extracellular LPA degradation and attenuation of LPA-induced neurite retraction after PRG1/LPPR4 overexpression in neuroblastoma cells, giving evidence for PRG1/LPPR4 being a potent modulator of phospholipid-mediated signaling.A single nucleotide polymorphism(SNP, rs138327459, NHLBI Exome Sequencing Project https://esp.gs.washington.edu/drupal/) was recently detected in humans resulting in an arginine(R) to threonine(T) exchange at position 345 of the amino acid chain of PRG1(mouse homolog located at position 346). To directly assess the potential functional consequenses of this human SNP in terms of their capacity to internalise LPA, I transfected primary hippocampal nuerons obtained from PRG1 deficient mouse either with PRG1/R346 T expressing construct(PCAGIG-GFP-PRG1-R346T) or PRG1-WT expressing construct(PCAGIG-GFP-PRG1-wt). Then made live imaging to recore the cellular uptake of extracellular LPA which was tagged by a fluorescence lable(Top Fluor, TF-LPA) with EpiFluorescence Microscope to analyze the internalisation of LPA. TF-LPA internalization capacity of either PRG-1wt or PRG-1R346 T was compared to PRG-1 deficient neurons in the same experiment. To rule out the dominant-negative effect of PRG1-R346 T, I used the same approach and transfected primary neurons obtained from wild type mice either with a PRG1-R346 T or a control GFP-construct. I also transfected primary neurons obtained from PRG1 deficient mouse with the same GFP construct to rule out the possible effect of GFP domain. The untransfected nuerons in the same experiment were used as control. Based on the statistics analysis result, while expression of PRG-1wt in neurons induced a significant increase in TF-LPA internalization,PRG-1R346 T expression failed to do so(pPRG1-WT VS PRG1-/-=0.001<0.05; p PRG1-R346 T VS PRG1-/-=0.361>0.05). And there are no alternation on LPA internalisation between PRG1-R346 T and GFP construct transfected neurons, neither in untransfected cells, which indicated that these studies confirmed that PRG-1R346 T was a loss-offunction mutation in a neuronal context. Moreover, I used Pitstop negative and pitstop positive, which are endocytocis inhibitor and agonist, to incubate the nuerons before live imaging, untreated cells are as control. The statistics result showed obvious big difference(N>20,PInhibitorvscontrol=0.18,PAgonistvscontrol=0.30), which suggests that the uptake function of the neurons is not via endocytosis mechanism. These data helps the further study of the function of PRG1 and LPA signaling, and provide effectable theoretical basis for the new therapy of the neurological disease induced by PRG1.