The Stimulatory Effects Of Low-dose Ionizing Radiation On Proliferation And Differentiation Of Adult Neural Stem Cells And Wnt/β-catenin Signaling Mechanism

PURPOSE OF STUDYParkinson’s disease (PD) is one common and severe neurological diseasethat characterizes with a selective degeneration of dopamine (DA) neurons inthe substantia nigra and disease progression, and at present it is still lack ofclinical cure method targeting on arresting neuronal progressive death.Growing evidences have shown that embryonic and adult neural stem cellswhich can potentially produce new neurons and neurotrophic factors in vivo andin vitro. Therefore, the cell replacement strategy by transplantation of neuralstem cells and inducement of dopaminergic neurons derived neural stem cells tosupply lost and decreased neurons is highly recommended for the preventionand treatment and of PD.In this project, by focusing on new intervention method or manipulatingmolecular target for cell therapy against PD， we have performed theseexperiments, i.e. comparison of midbrain and hippocampus-derived adult neuralstem cells in proliferation, cell survival, differentiation and neurotrophicproperties; stimulatory effects of low-dose radiation on proliferation anddifferentiation of adult neural stem cells and Wnt/β-catenin signalingmechanism, and dynamic changes of neurotrophic Trks signaling in the dopaminergic neurons of substance nigra in MPTP-induced PD model. Weexpect to find a new intervention method activating adult neural stem cells forcell therapy against PD in human beings.MATERIAL AND METHODS1．Cell culture of neural stem cells: The midbrain and hippocampus-derivedneural stem cells were prepared and cultured.2．Low dose ionizing radiation treatment: The midbrain and hippocampusNSCs were treated by X-ray ionizing radiation on which effected by linearaccelerator.3．Preparation of PD animal model: PD mouse model was prepared bysystemic MPTP injection in C57/BL mice.4．BrdU incorporation experiment: BrdU was incorporated into nucleus toshow cell division or proliferation state.5．Flowcytometry: Cell survival, necrosis and apoptosis of cultured neural stemcells were examined by flowcytometry analysis.6．Fluoro-Jade C staining: The neuronal degeneration in the substantia nigra ofMPTP model was visualized by Fluoro-Jade C staining.7．Western blot analysis: Western blot analysis were used to confirm expressionlevels of Wnts、BDNF and TrkB proteins.8．Immunocytochemistry and double labeling: Cell expression and changes ofWnts、BDNF and TrkB were performed by immunocytochemistry.9．Laser scanning confocal microscopy: Cellular localization pattern andchanges of Wnts、BDNF and TrkB were observed by laser scanning confocalmicroscopy. MAJOR RESULTS1． Comparison study of midbrain and hippocampus-derived neural stemcells in proliferation, survival, differentiation and neurotrophicproperties①Double immunofluorescence and laser scanning confocal microscopyconfirmed that nestin and BrdU was co-expressed in the cultured neuralstem cells. The midbrain-derived NSCs showed less or slowerproliferation than that of hippocampus-derived NSCs.②The midbrain-derived NSCs showed higher rate of survival and lowerrate of apoptosis in comparison with that of hippocampus NSCs byflowcytometry.③Differentiation into Tuj-1, TH, Nurr-1, and GFAP-positive cells weredetected in cell culture. Quantitative analysis indicated thatmidbrain-derived NSCs showed Tuj-1+commitment rate similar to thatof hippocampus NSCs, but higher rate of Nurr-1+and TH+cells thanthat of hippocampus NSCs. In contrast, however, hippocampus NSCsexhibited higher rate of GFAP+cell differentiation than that of midbrainNSCs.④Immunocytochemistry and Western blot showed expression of BDNF,GDNF, CDNF and DJ-1both midbrain NSCs and hippocampus NSCs.The midbrain NSCs and migrating cells exhibited strong BDNF andDJ-1staining, similar CDNF staining, and weaker GFAP staining incomparison with that of hippocampus NSCs.This result has indicated that midbrain neural stem cells show uniqueproperties in low proliferation, high neuronal commitment, and generation ofneurotrophic factors such as BDNF with therapeutic potential for PD. 2. Stimulatory effects of low-dose radiation on cell proliferation,differentiation of adult neural stem cells, and Wnt/β-catenin signalmediating mechanism①Flowcytometry was used to examine stimulating effects on proliferationof neural stem cells. Increasing of cell survival, proliferation anddecreasing of cell apoptosis were detected in low-dose radiation(0.3Gy) group, by comparing to that of control and high-dose radiation（3.0Gy）group（P<0.05vs control or high-dose group）②Immunocytochemistry showed that expression of Tuj-1or Tuj-1-positive cells was increased in low-dose radiation(0.3Gy) group, incomparison with that control and high-dose radiation（3.0Gy）group（P<0.05vs control or high-dose group）.③Western blot further indicated that expression of Wnt1,Wnt3a,Wnt5aand β-catenin were up-regulated in low-dose radiation (0.3Gy) group,in comparison with that of control and high dose radiation（3.0Gy）group（P<0.05vs control or high-dose group).④Wnt signaling block experiment showed that IWR1significantly inhibitstimulating effect on expression of Wnt1,Wnt3a,Wnt5a and β-cateninproteins after low-dose radiation by Western blot analysis.⑤Immunocytochemistry and confocal microscopy further confirmed thatIWR1could inhibit stimulating effect on proliferation, migration anddifferentiation of cultured neural stem cells after low-dose radiation.Under phase contrast microscopy, increasing of cell apoptosis,decreasing of proliferation of neural stem cells, and abilities ofneurospheres formations and migration and differentiation were clearly observed in0.3Gy+IWR1group.These results have indicated that low-dose radiation could stimulate cellproliferation, migration and differentiation of adult neural stem cells mostpossibly by triggering Wnt/β-catenin signaling pathway.3. Localization and changes of neurotrophic factor receptor TrkB and TrkCin dopaminergic neuron of substantia nigra in MPTP-mouse model①Double immunofluorescence and Western Blot analysis confirmed thatexpression of TrkB or TrkC were abundant in the substantia nigra,especially TrkB and TrkC-positive neurons were numerously observedin the substantia nigra pars compacta except of TrkA.②Western Blot analysis verified that down-regulation of TrkB and TrkCproteins expression were found in dopaminergic neurons of substancenigra of MPTP mouse model.③Though TrkB and TrkC positive cells were decreased in nigral neuronsin MPTP model, TrkB-positive survival neurons remained more incontrast to TrkC positive cells after MPTP insult. It showed that lostof TrkB positive cells is less than that of TrkC positive cells afterMPTP-insulted C57/BL mice.This result indicated that TrkB-positive dopaminergic neurons might beless sensitive to MPTP insult in the substantia nigra, suggesting thatBDNF-TrkB may crucially function in protection of dopaminergic neuronsunder PD state.SUMMARY AND CONCLUSIONSIn summary, we have obtained these important results in this study： 1．Midbrain-derived neural stem cells showed unique biological propertiesin low proliferation, high dopaminergic neurons commitment, andneurotrophic factor such as BDNF generation with therapeutic potentialfor PD.2．Low-dose radiation could stimulate cell proliferation, cell differentiationof midbrain-and hippocampus-derived neural stem cells, and theseeffects were most possibly mediated by triggering Wnt/β-cateninsignaling pathway.3．PD animal study indicated that TrkB-positive dopaminergic neuronswere less sensitive to MPTP insult in the substantia nigra of adult mice,suggesting that BDNF-TrkB might crucially act in neuroprotection ofdopaminergic neurons under PD state.In this study, we have studied and found new method that low-doseradiation can effective stimulate proliferation and differentiation of adult neuralstem cells possibly by Wnt/β-catenin signaling activation mechanism. Data ofthis study has thus provided important evidence for further investigation ofpractical cell therapy and molecular targeting strategy in treatment of PD.