| Parkinson’s disease (PD) is the second most common chronic neurodegenerativedisease in the population. Glial cell line-derived neurotrophic factor (GDNF) is suggestedas a specific neurotrophic factor for dopaminergic (DA) neural cells. GDNF is a strongcandidate agent which could prevent progressive degeneration of DA neural cells.However, heretofore, the molecular and cellular mechanism of GDNF is not fullyunderstood. In2003Paratcha et al. proposed that neural cell adhesion molecule (NCAM)was an alternative signaling receptor for GDNF. However, whether NCAM couldmediate the protective effect of GDNF on injured DA neural cells was not clear.In the present study, we try our best to explore the effect and the mechanism of NCAMon GDNF protecting DA neural cells. There are three parts in this study:I Differentiated SH-SYSY were treated with6-hydroxydopamine (6-OHDA) to builtinjured cellular model of DA neural cells.SH-SY5Y cells were treated with all trans-retinoic acid (ATRA) and brain derivedneurotrophic factor (BDNF) to sequentially differentiate into neural cells ofdopaminergic phenotype. Then differentiated SH-SY5Y cells were treated with6-OHDA to build cellular model of PD in vitro.1Morphologic variations of undifferentiated SH-SY5Y cells: undifferentiated SH-SY5Ycells had small neurites shorter than cell body. After being treated with ATRA and BDNF,cells were sequentially differentiated with neurites outgrowth to crosslink with each other.Differentiated cells maintain a neuronal morphology.2. The expression of neuron specific enolase (NSE) and tyrosine hydroxylase (TH) werevery low in undifferentiated SH-SY5Y cells. After differentiation, the NSE and THexpression significantly increased, indicating that SH-SY5Y cells have been differentiatedinto neurons with dopaminergic phenotype.3. Injures on differentiated SH-SY5Y cells by6-OHDA was in a dose-dependent fashion.Treatment of40μmol/L6-OHDA for24h could decrease the cell viability to50%. So insubsequent experiments, the concentration of40μmol/L was chosento treat cells.4. Low level of Cleaved caspase-3was detected in differentiated SH-SY5Y cells treatedwith40μmol/L for4h. The expression of Cleaved caspase-3increased gradually afterexposure to6-OHDA for6,8and10h. At time point of10h, the expression of Cleavedcaspase-3was at maximum (P<0.05), and in subsequent experiments we detected cellapoptosis at the time point of10h.The above results showed:SH-SY5Y cells treated with ATRA and BDNF were sequentially differentiated into neuralcells of dopaminergic phenotype.6-OHDA could injure the differentiated SH-SY5Y cells,decrease the cell viability and induce apoptosis of SH-SY5Y cells to increase theexpression of Cleaved caspase-3.II NCAM could mediate the protective effect of GDNF on injured DA neural cellsRecently, it was found that NCAM was an alternative signaling receptor for GDNF andcould medicate GDNF signaling pathways involved in cell proliferation, differentiation,migration and neuritis growth. However, whether NCAM could mediate the protectiveeffect of GDNF on injured DA neural cells is not clear. In order to find out the underlyingmechanism, we constructed NCAM short hairpin RNA (shRNA) plasmid and NCAMplasmid as lentiviral (LV) vectors to suppress and promote NCAM expression respectively, and detected cell viability with Cell Counting Kit-8, cell apoptosis with flow-cytometry,and expression of Cleaved caspase-3with Western blot, to observe whether NCAM couldmediate the protective effect of GDNF on injured DA neural cells by6-OHDA.1. The results of Western blot indicated that NCAM-140was the main subtype indifferentiated SH-SY5Y cells, while NCAM-180and NCAM-120were of low expression.2. NCAM shRNA plasmid and NCAM plasmid as the LV vectors were constructedsuccessfully. The results of polymerase chain reaction (PCR) and Western blot indicatedthat NCAM-shRNA3and NCAM LV vector could suppress and promote NCAMexpression at the level of mRNA and protein respectively.3. GDNF could protect6-OHDA injured differentiated SH-SY5Y cells in a dose dependedfashion. At the concentration of50,100, and200ng/ml, GDNF could increase cellviability to75.48%±4.2%,78.98%±4.47%and80.37%±4.49%of the normal control.In order to be cost-effective, we chose the concentration of50ng/ml in subsequentexperiments.4. Overexpression or suppressed NCAM expression could influence protective effect ofGDNF.4.1Cell viability was reduced to45.77±3.87%in SH-SY5Ycells treated with40μmol/L6-OHDA for24h. Treatment with GDNF could increase cell viability to70.34±3.69%.Compared to the6-OHDA group, the difference was statistically significant (P<0.05),indicating that GDNF could protect6-OHDA-injured SH-SY5Y cells. The cell viabilityincreased to87.45±4.77%in the NCAM+6-OHDA+GDNF group. Compared to the6-OHDA+GDNF group, the difference was statistically significant (P<0.05). This resultindicated that overexpression of NCAM promoted the protective effect of GDNF. The cellviability in the shRNA+6-OHDA+GDNF group was58.95±2.91%. Compared to the6-OHDA+GDNF group, the cell viability increased and the difference was statisticallysignificant (P<0.05). Compared to the6-OHDA group, the cell viability decreased and thedifference was statistically significant (P<0.05). These results indicated that decreasedNCAM expression could partially suppress protective effect of GDNF on6-OHDA-injured cells. 4.2The low apoptotic rate was detected when the cells were not injured. The apoptoticrate improved to30.67±1.96%in SH-SY5Ycells treated with40μmol/L6-OHDA for10h. Treatment with GDNF could decrease the rate of apoptosis to17.48%±1.98%.Compared to the6-OHDA group, the difference was statistically significant (P<0.05),indicating that GDNF could suppress6-OHDA induced apoptosis. The apoptotic ratedecreased to12.67±0.99%in the NCAM+6-OHDA+GDNF group. Compared to the6-OHDA+GDNF group, the difference was statistically significant (P<0.05). This resultindicated that overexpression of NCAM promoted the protective effect of GDNF. Theapoptotic rate in the shRNA+6-OHDA+GDNF group was22.98±1.28%. Compared to6-OHDA+GDNF group, the apoptotic rate increased and the difference was statisticallysignificant (P<0.05). Compared to the6-OHDA group, the apoptotic rate decreased andthe difference was statistically significant (P<0.05). These results indicated that decreasedNCAM expression could partially suppress protective effect of GDNF on6-OHDA-injured cells.4.3No expression of Cleaved caspase-3was detected when cells were not injured. Theexpression of Cleaved caspase-3was improved to0.88±0.06in SH-SY5Y cells treatedwith40μmol/L6-OHDA for10h. Treatment with GDNF could obviously decrease theexpression of Cleaved caspase-3to0.49±0.05. Compared to the6-OHDA group, thedifference was statistically significant (P<0.05), indicating that GDNF could suppress6-OHDA-induced expression of Cleaved caspase-3. The expression of Cleaved caspase-3decreased to0.31±0.04in the NCAM+6-OHDA+GDNF group. Compared to the6-OHDA+GDNF group, the difference was statistically significant (P<0.05). This resultindicated that overexpression of NCAM promoted the protective effect of GDNF. Theexpression of Cleaved caspase-3in shRNA+6-OHDA+GDNF group was0.42±0.03.Compared to6-OHDA+GDNF group, the expression of Cleaved caspase-3increased andthe difference was statistically significant (P<0.05). Compared to the6-OHDA group, theexpression of Cleaved caspase-3decreased and the difference was statistically significant(P<0.05). These results indicated that decreased NCAM expression could partiallysuppress protective effect of GDNF on6-OHDA induced apoptosis. The above results showed:GDNF could improve cell viability, decrease the apoptotic rate and expression ofCleaved caspase-3in6-OHDA-injured SH-SY5Y cells. Overexpression of NCAM couldfurther increase cell viability, decrease apoptotic rate and expression of Cleaved caspase-3.So overexpression of NCAM promoted the protective effect of GDNF. Suppressed NCAMexpression decreased cell viability, increased the apoptotic rate and expression of Cleavedcaspase-3, and partially suppressed protective effect of GDNF on6-OHDA-inducedapoptosis.III The role of lipid rafts in NCAM medicated protective effect of GDNF on DAneural cellsLipid rafts are the microdomains in the cell membrane, floating freely, enriched incertain lipids cholesterol and sphingomyelin. It plays an important role in NCAMsignaling transduction. But it is unclear whether lipid rafts are involved in NCAMmedicated protective effect of GDNF on DA neural cells. Nonionic detergent Triton X-100and OptiPrep density gradient medium were used to isolate lipid rafts and the effect ofGDNF on NCAM distribution in lipid rafts was observed.2-bromopalmitate (2-BP) wasused to suppress palmitoylation of NCAM, and NCAM translocation to lipid rafts wasabolished. Whether lipid rafts involved in NCAM medicated protective effect of GDNF onDA neural cells was observed1. GDNF could induce NCAM translocation to lipid raftsWhen nonionic detergent Triton X-100was used to isolate lipid rafts, it was found that11.08±0.69%of NCAM located in lipid rafts in SH-SY5Y cells without GDNFtreatment. After GDNF treatment, NCAM located in lipid rafts increased gradually. Atthe time point of15min, NCAM located in lipid rafts reached the maximum (40.21±2.34%).When OptiPrep density gradient medium was used to isolate lipid rafts, it was foundthat9.05±0.89%of NCAM located in lipid rafts in SH-SY5Y cells without GDNFtreatment. After GDNF treatment for15min, NCAM located in lipid rafts increased to30.47±2.98%. This result was consistent with these results obtained in the experiments using nonionic detergent.2.2-BP could suppress the translocation of NCAM to lipid rafts induced by GDNF.Pretreated with20μM2-BP for30min decreased the amount of NCAM in lipid rafts(17.25±1.02%). Compared to the GDNF group, the difference was statistically significant(P<0.05).3. Pretreatment with2-BP suppressed NCAM medicated protective effect of GDNF.3.1The cell viability in the2-BP+6-OHDA+GDNF group decreased to60.3±4.36%.Compared to the6-OHDA+GDNF group, the cell viability decreased and the differencewas statistically significant (P<0.05). Compared to the6-OHDA group, the cell viabilityincreased and the difference was statistically significant (P<0.05). These resultssuggested that2-BP could partially suppress the protective effect of GDNF on6-OHDA-injured DA neural cells. The cell viability in NCAM+2BP+6-OHDA+GDNF group was61.59±3.91%. Compared to the NCAM+6-OHDA+GDNF group,cell viability decreased with statistical significance (P<0.05). Compared to the shRNA+6-OHDA+GDNF group, cell viability in the NCAM+2-BP+6-OHDA+GDNF groupdecreased, but without statistical significance (P>0.05).3.2The apoptotic rate in the2-BP+6-OHDA+GDNF group was22.83±1.09%.Compared to the6-OHDA+GDNF group, the apoptotic rate increased and the differencewas statistically significant (P<0.05). Compared to the6-OHDA group, the apoptotic ratedecreased and the difference was statistically significant (P<0.05). These resultssuggested that2-BP could partially suppress the protective effect of GDNF on6-OHDA-injured DA neural cells. The apoptotic rate in the NCAM+2-BP+6-OHDA+GDNF group was21.69±1.39%. Compared to the NCAM+6-OHDA+GDNF group,apoptotic rate increased with statistical significance (P<0.05); Compared to the shRNA+6-OHDA+GDNF group, apoptotic rate in the NCAM+2BP+6-OHDA+GDNF groupwas not statistically different (P>0.05).3.3The relative expression level of Cleaved caspase-3in the2-BP+6-OHDA+GDNFgroup was0.79±0.05. Compared to the6-OHDA+GDNF group, the expression ofCleaved caspase-3increased and the difference was statistically significant (P<0.05). Compared to the6-OHDA group, the expression of Cleaved caspase-3decreased and thedifference was statistically significant (P<0.05). These results suggested that2-BP couldpartially suppress the protective effect of GDNF on6-OHDA injured DA neural cells bysuppress the translocation of NCAM to lipid rafts. The expression of Cleaved caspase-3in the NCAM+2-BP+6-OHDA+GDNF group was0.77±0.06. Compared to theNCAM+6-OHDA+GDNF group, expression of Cleaved caspase-3increased withstatistical significance (P<0.05); Compared to the shRNA+6-OHDA+GDNF group,expression of Cleaved caspase-3in the NCAM+2-BP+6-OHDA+GDNF group wasnot statistically different (P>0.05).The above results showed:NCAM existed in lipid rafts in SH-SY5Y cells without GDNF treatment. After GDNFtreatment, NCAM located in lipid rafts increased. At the time point of15min, NCAMlocated in lipid rafts reached the maximum.2-BP could suppress palmitoylation ofNCAM to suppress the translocation of NCAM to lipid rafts induced by GDNF and italso could suppress GDNF-NCAM protecting DA neural cells. These results suggestedthat translocation of NCAM to lipid rafts in the premise to mediate the function of GDNFprotecting DA neural cells, and lipid rafts played a key role in the protective effect ofGDNF mediated by NCAM.The present study aims to explore whether NCAM, a novel GDNF receptor,participates in protective effect of GDNF on DA neural cells, and the effect oftranslocation of NCAM to lipid rafts during the signal transduction. These results wouldcontribute to the understanding of the protective effect of GDNF on DA neural cells andto the prevention and treatment of PD. |
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