The Role And Mechanism Of Parathyroid Hormone Related Peptide Nuclear Localization Sequence And C-terminus In Regulating Oligodendrocytes Development

To observe the expression of parathyroid hormone related peptide(PTHrP)and its receptor(PTHrPR)in the spinal cord and oligodendrocytes(OLs),we used immunohistochemistry to detect the expression of PTHrP and its receptor in the spinal cord.Results showed that PTHrP and its receptor was expresssed in the spinal cord from postnatal days(P)3,7 and 14 C57B1/6 mice,and the expression of PTHrP in white matter was stronger than that in gray matter.Myelin basic protein(MBP)and PTHrP double immunofluorescence staining displayed that PTHrP was expressed in the mature oligodendrocytes from the spinal cord of P14 mouse.With the means of magnetic activated cell sorting,we sorted 04-positive oligodendrocyte precursor cells(OPCs)from the cortex of P3 C57B1/6 mouse and cultured them in vitro.After grown for 7 days,cells were labeled with PTHrP immunofluorescence staining.Results showed that PTHrP was expressed in 04-positive OPCs.These results suggest that PTHrP may play an important role in the development of oligodendrocytes.PTHrP Knock-In(KI)mice was used to study the effect of deficiency of PTHrP nuclear localization sequence(NLS)and C-terminus on the OLs development.This animal model was generated by introducing the aminoacid translation termination codon TGA into 84 aminoacid of PTHrP gene and expressed only PTHrP1-84,and lacking the NLS and C-terminus.We used techniques of histology,cellular and molecular biology to analyze the phenotypic difference of OLs between P7 or P14 wild type(WT)and KI mice through in vivo and in vitro experiments.For in vivo study,we observed the changes of OLs development in PTHrP KI mice compared with which in WT mice.The results of immunofluorescence staining of Olig2 as the marker for OPCs,and MBP as the mature OLs’s marker showed that the percentage of Olig2 or MBP positive cells was significantly reduced in the brain and cervical spinal cord segment of P7 and P14 KI mouse.The results of real-time quantitative RT-PCR showed that Olig2,CNPase,MBP mRNA expression levels were significantly downregulated in the brain and spinal cord of P7 and P14 KI mouse.The results of western blot showed that PDGFRa and Olig2 expression levels were downregulated in the spinal cord of P7 and P14 KI mouse.These results indicate that the deficiency of PTHrP NLS and C-terminus in the brain and spinal cord may delay the OLs development.To clarify whether the deficiency of PTHrP NLS and C-terminus in CNS of mice lead to the delay of OLs development was related to the decrease of cellproliferation,we injected BrdU into P7 and P14 WT and KI mouse and used BrdU/Olig2 double immunofluorescence staining to analyse the differences of numbers of proliferating OPCs in PTHrP KI mice and their WT littermates.The results showed that the number of BrdU/Olig2 positive cells in the ventrolateral cervical spinal or cerebellum of P7 and P14 KI mice was less than that of WT mice,and the number of BrdU/Olig2 double positive cells in hippocampus of P7 KI mice was less than that of WT mice.These results indicate that the deficiency of PTHrP NLS and C-terminus may inhibit the OPCs proliferation and delay OLs development.The results from this study demonstrated that the development of OLs was delayed in the CNS of PTHrP KI mice.It may resulted from the direct effect of deficiency of PTHrP NLS and C-terminus on OLs,or from the changes of the micro-environment surrounding the OLs in the CNS of PTHrP KI mice and effect on the OLs indirectly.To further clarify direct effect of the deficiency of PTHrP NLS and C-terminus on OLs development,we used the 04-positive magnetic activated cell sorting method to sort OPCs from the cortex of P3 WT and KI mice,and cultured in vitro to observe the changes of OPCs proliferation,apoptosis,differentiation,indexes related cell cycle and cellular oxidative stress.04 positive OPCs were sorted from WT and KI mice and were cultured for 9 days and continued with the differention cultures for another 3 days,then cells were fixed and labeled with CNPase,APC(immature OLs marker),or MBP immunofluorescence staining to observe the direct effect of the deficiency of PTHrP NLS and C-terminus on OPCs differentiation and maturation in vitro.The results showed that the number of CNPase and APC positive cells from KI mice were reduced.The cytoplasm was pale and dendrites were reduced in these cells from the KI mice compared with those from WT mice.There were no MBP positive cells in the cultured cells from the KI mice.These in vitro results indicate that the deficiency of PTHrP NLS and C-terminus can directly lead to the disorder of OPCs differentiation and maturation.The mRNA and proteins were isolated from primary cultured OPCs and the differentiation-induced OPCs and were analyzed used real-time quantitative RT-PCR and Western blots.The results showed that,compared with the primary OPCs from WT mice,Olig2 and CNPase mRNA expression was downregulated,PDGFRa,Olig2,Rip protein expression levels were downregulated,Bmil and Bcl2 mRNA expression levels were downregulated,Bmil,PCNA protein expression levels were also downregulated,while the protein expression levels of Caspase3 markered for apoptosis were upregulated,p19,p21,p27,p53 mRNA expression levels were significantly upregulated,CDK2 and CyclinE mRNA expression levels were significantly downregulated,and p16 protein expression was upregulated,mRNA expression levels of antioxidant enzymes SOD 1,GPX1,CAT were downregulated in the primary OPCs from KI mice.In differentiated OPCs after 7 days’ differentiation cultures from KI mice,Olig2,CNPase,MBP,Bcl2 and Bmil mRNA expression levels were significantly downregulated,whereas p19,p21,p27,p53 mRNA expression levels were significantly upregulated,CDK2 and Cyclin E mRNA expression levels were significantly downregulated,and SOD1,GPX1 and CAT mRNA expression levels were significantly downregulated.The in vitro results demonstrated that the deficiency of PTHrP NLS and C-terminus could directly lead to OLs developmental disorders,which were associated with the decreased proliferation and differentiation,reduced antioxidant capacity,and increased apoptosis of OPCs.In summary,the results of this study identify that the deficiency of PTHrP NLS and C-terminus can inhibit OPCs proliferation,increase apoptosis and delay OLs development by upregulating the expression levels of p16,p19,p21,p27,p53,downregulating the expression levels of CDK2 and Cyclin E.in addition,the deficiency of PTHrP NLS and C terminus also decreases OPCs antioxidant capacity,increase oxidative damage and apoptosis,and lead to the disorder of OLs development.The results of this study preliminarly clarify the role of PTHrP NLS and C-terminus on the OLs development,and provide the preliminary experimental basis for developing the PTHrP NLS and C-terminus as a new therapeutic target for CNS demyelinating diseases.