A Study On Mechanism Of Action Of Parathyroid Hormone Related Peptide Nuclear Localization Sequence And Carboxyl Terminus On Central Nervous System Development

To determine whether the effect of deficiency of the parathyroid hormone related peptide (PTHrP) nuclear localization sequence (NLS) and C-terminus on brain development was associated with reduction of the self-renewal ability of neural stem cells (NSCs), NSCs derived from hippocampus of3-day-old PTHrP KI mice (i.e., PTHrP NLS and C-terminus deficient mice) and their wild-type (WT) littermates were cultured primarily or secondarily to form neurospheres and analysed by computer assist analysis, the proliferation and apoptosis of NSCs were assessed using techniques of BrdU incorporation, propidium iodide (PI) staining or double staining with PI and Annexin V, flow cytometry and Western blot. Results showed that the neurosphere forming efficiency in primary or secondary cultures and ratio of neurosphere formation in the secondary cultures relative to the primary cultures were reduced significantly; The proliferation of NSCs was decreased, while the apoptosis of NSCs was increased; the expression levels of CDK2and CDK4were down-regulated, while the expression levels of p16, p21, p27and caspase-3were upregulated in NSCs from PTHrP KI mice compared with those from WT mice. These results indicate that PTHrP NLS and C-terminus play an important role in maintaining NSC self-renewal through activating CDK2and CDK4and inhibiting pl6, p21and p27to stimulate the proliferation and to inhibit the apoptosis of NSCs.To assess the effect of PTHrP NLS and C-terminus deficiency on spinal cord development, phenotypes of spinal cord were compared between1-or2-week-old PTHrP KI mice and WT mice using histological, immunohistochemical and molecular biological approaches. Results showed that the length of spinal cord was shorter, the weight and cross section area of the spinal cord were reduced; The percentages of NeuN positive neurons and myelin basic protein (MBP) positive oligodendrocyte areas and the number of myelin were reduced dramatically in spinal cords from PTHrP KI compared with those from WT mice. The percentage of the proliferating cell nuclear antigen (PCNA) positive cells was decreased significantly; the expression of Cyclin D1, CDK4and CDK6at mRNA levels and of Cyclin D1, Cyclin E, CDK4and CDK6at protein levels were downregulated significantly, whereas the expression of p16, p19, p21and p27at both mRNA and protein levels were upregulated in spinal cord from PTHrP KI mice compared with WT mice. These results indicate that PTHrP NLS and C-terminus play an important role in stimulating spinal cord development by stimulating NSC proliferation and differentiation into neurons and oligodendrocytes and accelerating myelin formation.To determine whether p27as one of downstream targets of PTHrP NLS and C-terminus is involved in the regulation in brain development, we generated compound mutant mice which are homozygous for both p27deletion and the Pthrp KI mutation (p27-/-Pthrp KI), and compared p27-/-Pthrp KI mice with p27-/-, Pthrp KI, and WT littermates at2weeks of age using techniques of histology, immunohistochemistry and Western blot. Results showed that Deletion of p27Kip1in Pthrp KI mice resulted in a longer lifespan, increased body weight and improvement in the morphology of brain, although Pthrp KI mice were not normalized. The protein expression levels of Cyclin D1were up-regulated, whereas the the protein expression levels of p19, p21and p53were down-regulated, the proliferation of neural cells and GFAP and MBP positive areas were increased significantly in p27-/-Pthrp KI mice compared with Pthrp KI mice. The protein expression levels of sonic hedgehog (Shh) pathway related molecules including including Shh, Smoothened (Smo), Gli1/2and Bmi1were up-regulated in brain tissue of p27-/-mice, and down-regulated in Pthrp KI mice compared with their WT littermates, however, these parameters were up-regulated in p27-/-Pthrp KI mice compared with Pthrp KI mice. These results indicate that deletion of p27can partially rescue defects caused by the deficiency of PTHrP NLS and C-terminus through activating Shh pathway and stimulating NSC proliferation and differentiation into neuron and glial cells.Results from this study were not only revealed the mechanism of PTHrP NLS and C-terminus in regulating CNS development, but also provided experimental and theorical evidence for clinical application of PTHrP NLS and C-terminus in promoting nerve regeneration.