The Mechanism Of PLKs Mediate Cell Cycle Arrest And Prp^Sc Elimination In Central Neuronal CELL

Prion is a kind of infectious protein, which can infect the central nervous system of human and many other mammalian. Prion related diseases include CJD, kuru and GSS syndrome in humans, scrapie infected with sheep, BSE in infected with cattle. It is commonly accepted that this kind of diseases is associated with the conversion of cellular prion protein (PrPc) into an abnormal conformer (PrPSc) with unknown factors. The pathogenic PrPSc is insoluble and infectious. In the early stage of disease, the abnormal folding of proteins can cause oxidative stress and induce cell injury, in order to protect neuron cells from death, autophage or apoptosis related signaling may be activated. In the late stage of disease, large numbers of PrPSc proteins cannot be cleared, resulting in deposition of neurotoxic protein. The hallmarks of TSE are characterized with neuronal loss, spongiform degeneration, reactive gliosis and PrPSc amyloid plaque formation in brain tissue.Although we have got some information about the neuronal death in prion disease, its mechanism has not been elucidated in detail. For a long time, mature neuron is believed to be terminal differentiation cell and can not divide. While growing evidences are arising for the abnormal expression of cell cycle related protein in several neurodegenerative diseases, including Alzheimer’s disease (AD) and Parkinson’s disease (PD). One may propose that terminal differentiation neuron can reenter to the cell cycle under some irritation. Polo-like kinases (PLKs) consist of a kinase family expressed in all embryonic tissues, which play critical role during multiple stages of cell cycle progression, including centrosome maturation, mitotic entry, chromosome segregation. It has been generally accepted that the increase of PLK1 and decrease of PLK3 are associated with the developments and metastases of several human malignant tumors. In addition to participate in the regulation of cell cycle, PLK2/3 is also involved in the regulation of synaptic plasticity in the central nervous system.Since the cell cycle reentry events exist in prion disease, and PLK1/3 play a key role in regulating G2/M phase transformation process in the cell cycle. Besides, our previous gene chip transcription analysis found the expression of PLK1 increased and PLK3 decreased in brain tissues of prion disease, so we proposed that PLKs involved in abortive cell cycle events, and eventually lead to neuronal apoptosis in prion disease. In this research, we use prion infection cell model and animal models by Immunohistochemistry and Western blot techniques to investigate the function and mechanism of PLKs mediated abortive cell cycle events and PrPSc elimination in prion disease, which can provide laboratory basis for the accurately clinical treatment of prion disease.Part one:PLK1/3 mediated abortive neuronal cell cycle events in prion disease.In order to observe whether PLK participate the regulation of neuronal cell cycle reentry, the alterations of the PLK1 and PLK3 in brains were evaluated in the current study using the hamster model infected with scrapie. Through Western blots, immunohistochemical and immunofluorescent assays, remarkably increase of PLK1 and decrease of PLK3 were observed in the brains of scrapie strain 263K-infected hamsters, dynamic analysis observed the alteration of PLK at the early stage of infection. Both PLK1 and PLK3 were observed in neurons but not in astrocyte cells by confocal microscopy, thus the PLKs alteration took place in neuron. Accompanying with the changes of PLKs in the brains of 263K-infected hamsters, Cdc25C and its phosphorylated forms (Serl98 and Ser216) were significantly down-regulated, whereas Cyclin B1 and PCNA were obviously up-regulated, meanwhile, phospho-histone H3 remained almost unchanged. Similar alterations of PLKs were also detected in a scrapie infectious cell line SMB-S15. Moreover, in order to eliminate the influence of immortalized cells on cell cycle study, primary cortical neuron cells were cultured and exposure of the cytotoxic peptide PrP106-126, which induced similar changes as before.Part two:Polo-like kinase 3 (PLK3) mediates the clearance of the abnormal PrP.To further demonstrate the association of PLKs with prion disease, we confirmed the interaction between PLK3 and PrP protein both in vitro and in vivo by GST pull-down and immunoprecipitation. Both the kinase domain and polo-box domain of PLK3 were proved to bind PrP proteins expressed in mammalian cell lines. To delineate the biological significance between PLK3 and prion protein, different amounts of PLK3 and a fixed amounts of wide-type or mutant PrP were transfected to HEK293 cells. Overexpression of PLK3 did not influence the expression of wild-type PrP, but apparently decreased the levels of mutated PrPs in cultured cells. The kinase domain appeared to be responsible for the clearance of abnormally aggregated PrPs, but this function seemed not rely on its kinase activity. RNA-mediated knockdown of PLK3 obviously aggravated the accumulation of cytosolic PrP. After using ubiquitin-proteasome system and autophagy-lysosome system inhibitors, we found that ubiquitin-proteasome and macroautophagy pathway inhibitor failed to induce accumulation of the mutated PrP protein, while inhibition of lysosome obviously increased the aggravated PrP protein, which suggest that PLK3-mediated degradation of mutated PrP seems dependent on lysosomal mechanism. Moreover, PLK3-overexpression in a scrapie infected cell line caused notable reduce of PrPSc level, but had minimal effect on the expression of PrPc in its normal partner cell line.In this study, we demonstrated that as a response to abnormal irritations, neurons quickly attempt to reenter cell cycle by altering cell cycle machinery. However, possibly due to the ill-regulation for some key events in cell cycle during prion infection and neurons are short of the cell cycle-related components to accomplish mitosis, the cell cycle reentry is definitely abortive, which is connected with apoptosis of neurons with end-stage neuropathology. On the other hand, we confirmed the KD region of PLK3 responsible for the degradation of abnormal PrP. We therefore assume that the recovery of PLK3 in the early-stage of prion infection may be helpful to prevent the toxic accumulation of PrPSc and cell cycle reentry in the brain tissues, meanwhile, it also provides clues to other neurodegenerative diseases treatment.