Disinhibition Of Neuronal Neurite Outgrowth In The Presence Of Nogo-66 By SiRNA Mediated Knockdown Of NgR Of Neural Stem Cells

Disinhibition of neuronal neur ite outgrowth in the presence of Nogo-66 by siRNA mediated knockdown of NgR of neural stem cellsObjectivesSeveral proteins of CNS myelin possess axon growth- inhibiting properties, notably Nogo-A, myelin- associated glycoprotein (MAG), and oligodendrocyte myelin glycoprotein (OMgp). The 66 aa surface loop of Nogo is inhibitory for axonal growth in culture and acts via a Nogo-66 Receptor (NgR) expressed by neurons and localized to their axons in vitro. Remarkably, the NgR protein mediates the inhibitory action of MAG and OMgp, as well as Nogo-66. NgR is a target for treatment to promote axon recovery because Nogo, MAG, and OMgp all bind to NgR to mediate their inhibitory effects on neurite outgrowth.Exclude approaches aimed at disinhibiting axon outgrowth to treat spinal injury, regenerative medicine using stem cell biology is attracting a lot of attention because this is considered a particularly important therapeutic strategy for regeneration of the CNS. But if NSCs express NgR and could be affected by myelin associated inhibitory proteins in the progress of differentiation has not been proved.Nogo-P4 is an active segment of Nogo-66 and has the core inhibitory activity of Nogo-66. Here, we use immunocytochemistry and Western blot methods to detect NgR in NSCs derived from spinal cord of rat, compare the average neuronal neurite length,differentiated neuron percentage and percentage of neurite possessing neuron during the differentiation of NSCs with and without Nogo-P4, before and after siRNA-mediated knockdown of NgR on NSCs. Experimental methods1. AnimalsPostnatal 1 day Wistar rats were supplied by animal center of China Medical University.2. Main reagentsMonoclonal NgR antibody (Chemicon) was used to label NSCs at 10μg/ml for immunocytochemistry(ICC) and Western blots. Polyclonal neuron specific enolase (NSE) antibody, glial fibrillary acidic protein (GFAP) antibody and myelin basic protein (MBP) antibody were purchased from Boster Company and were used to label neurons and neurites, astrocytes and oligodendrocytes respectively at 1:300. Monoclonal nestin antibody (Chemicon) was used to label NSCs at 1:200. Second antibody Alexa Fluor(?) 488 was purchased from Invitrogen. PI(Sigma) was used for counterstaining to identify nuclei at 1:3000. DMEM/F12 medium,B27,PI,Block-It Fluorescent Oligo,LIPOFECTAMINE 2000 REAGENT and OPTI MEM were purchased from Invitrogen company.3. Culture and identification of NSCs3.1 Culture of NSCsThe culture methods were based on those described previously. Spinal cords of P1(postnatal 1 day) Wistar rats(animal center of China Medical University) were dissected and dissociated into single cells using a solution of 0.125% trypsin for 45min at 37℃. The suspension was centrifuged at 1000 rpm for 10 min at 4℃. Decant as much of the supernatant as possible. Resuspend the cells in a total volume of 10 mL of DMEM/F12 (Invitrogen) serum-free stem cell medium containing freshly added epidermal growth factor (EGF) (Chemicon) 20ng/ml, basal fibroblast growth factor(bFGF) (Invitrogen) 20ng/ml and B27((Invitrogen, 1:50) (prewarmed to 37℃). Cell suspension was cultured in a atmosphere containing 5% CO₂ at 37℃. 3.2 Expression of nestinNeurospheres in primary cultures or subcultures were collected by centrifugation at 1000 rpm for 10 min at 4℃. These spheres were fixed in acetone for 15 min after brief washes in 0.01M phosphatebuffered saline (PBS). Some were dropped on poly-d-lysine coated coverslips and immunostained intact to detect nestin immunoreactivity of the spheres.3.3 Differentiation of NSCsNSCs suspension was added to 6 well culture plate with glass cover slips pre-coated with poly-D-lysine in it and cultured in the presence of 10% FBS to initiate differentiation. After 1 week of differentiation cover slips were taken out for immunocytochemisty assay. The differentiated cells were incubated with primary antibodies against GFAP, MBP and NSE, respectively, and then exposed to secondary antibodies.Microscopic images of the immunostained neurospheres and differentiated cells were captured using a SpotRT digital camera (Diagnostic Instruments Inc.) mounted on an Olympus BX60 microscope (Olympus Optical Co.).4. Detection of NgR expressionNeurospheres in primary cultures or subcultures were collected by centrifugation at 1000 rpm for 10 min at 4℃. These spheres were fixed in acetone for 15 min after brief washes in 0.01M phosphatebuffered saline (PBS). Some were dropped on poly-d-lysine coated coverslips and immunostained intact to detect NgR immunoreactivity of the spheres.5. Design and synthesis of siRNASiRNA directed against NgR was designed and synthesized by Invitrogen Company. The siRNA sequences used were:siRNAASequence (5′to 3′): - UUU GAG UGC AGC CAC AGG AUG GUG A Sequence (5′to 3′): - UCA CCA UCC UGU GGC UGC ACU CAAAsiRNA BSequence (5′to 3′): - UCA GUG AGC UGA UUG GUC UGG AAG GSequence (5′to 3′): - CCU UCC AGA CCAAUC AGC UCA CUG AsiRNA CSequence (5′to 3′): - UGC AGU ACC UCU ACC UAC AAG ACAASequence(5′to 3′): - UUG UCU UGU AGG UAG AGG UAC UGC A6. GroupNSCs were divided into control group, Nogo-P4 group, siRNA group and siRNA+Nogo-P4 group. In control group, NSCs differentiated in the presence of 10% FBS; in Nogo-P4 group NSCs differentiated in the presence of 10% FBS and 4μmol/l Nogo-P4; in siRNA group, NSCs were transfected with siRNA before they differentiated in the presence of 10% FBS; in siRNA+Nogo-P4 group, NSCs were transfected with siRNA before they differentiated in the presence of 10% FBS and 4μmol/l Nogo-P4.7. SiRNA preparation and transfectionBefore transfection the neurospheres were triturated into single cells as much as possible. In one tube, 50pmol of siRNA was mixed in 50μl Opti-MEM, while a second tube contained 1μl Lipofectamine 2000 reagent and 50μl Opti-MEM and each was incubated at room temperature for 15min before the two solutions were combined and incubate for complex formation. Then the complex was added to NSCs suspension without antibiotics. After 1d, 3d and 5d the NSCs were collected and immunostained with NgR antibody to detect the knockdown efficiency.Block-It Fluorescent Oligo was fluorescence labeled duplex siRNA used for observation of transfection efficiency. The transfection procedures are as described above.8. Detection of knockdown efficiency 8.1 ImmunohistochemistryAfter 1d, 3d and 5d of transfection, the NSCs were collected and immunostained with NgR antibody to detect the knockdown efficiency. The cells attached to coverslips in differential cultures were briefly riNSEd with 0.01M PBS and fixed in 4% paraformaldehyde in 0.1M phosphate buffer for 30 min at 4℃.Before staining, cover slips were incubated sequentially in 0.3% Triton X-100 in PBS (PBS-T; 30 min), 10% goat serum in PBS (30 min), and then with primary antibodies for 24hr at 4℃. After incubating in the primary antibodies for 24hr, sections were washed in PBS and then incubated for 2 hr at room temperature with second antibody (Alexa Fluor(?) 488, Invitrogen).8.2 Western blotsThe NgR protein expression was detected by Western blot method. Cells were collected by centrifugation(1000rpm, 5min) and washed three times with ice-cold PBS, then lysed in buffer(50mM Tris-HCl, pH8.0, 150mM NaCl, 100μg/ml PMSF, 1%TritonX-100) for 30 rain at ice. After removal of cell debris by centrifugation (12, 000rpm, 4℃), the protein concentration of lysates was meatured by Bradford method, 40μg of each lysate sample was boiled for 5 min in sample buffer and were separated by 10% SDS-PAGE and transferred onto nitrocellulose membrane (Pall Corporation). Nonspecific reactivity was blocked in 5% nonfat dry milk in TBST (10mM Tris-HCl, pH7.5, 150mM NaCl, 0.05%Tween-20) for 1h at room temperature. The membrane was then incubated overnight at 4℃with monoclonal anti-NgR antibody (Chemicon) followed by reaction with alkaline phosphatase conjugated second antibody. Reactive protein was detected by Protein Detector BCIP/NBT Western Blotting kit (KPL).9. Measurement of neurite outgrowthNSE was used to label the neuron by immunocytoflurescence method. Neurite length was measured by Image-Pro Plus 5.0 software. 10. Statistical analysisThe numbers of NgR⁺ cells were determined in at least three samples in each culture. The total cell number was determined by PI counterstaining. About 400 cells of each culture were counted. One-Way Analysis of Variance was used for the comparison of different cultures. All the data were treated by SPSS 11.5 software.Results1. Expression of NgR on NSOsGrowth of neurospheres derived from spinal cord of rat is relatively slow. Neurospheres could be seen after suspension culture for 1 week and obtained in large amount after 2 weeks. The neurospheres express nestin positively and they could differentiate into NSE, GFAP and MBP positive cells after mitogens were removed and they were cultured in the presence of 10% fetal bovine serum. So the neurospheres are capable to generate neurons, astrocytes as well as oligodendrocytes and they have pluripotency.Both neurospheres and single neural stem cells express NgR positively after they were treated by immunocytochemistry.In the test of Western blot, NSCs were confirmed to express NgR apparently.2. Inhibition effect of Nogo-P4In Nogo-P4 group, Nogo-P4 was added to the culture medium during the differentiation progress of NSCs at the concentration of 4μmol/L. In control group, NSCs differentiated without Nogo-P4. In both Nogo-P4 group and control group, 10% FBS was added to the culture to initiate and promote differentiation. NSE was used to mark the neuron by immunocytofluorecence method. Neurite length of neurons differentiated from NSCs was measured under fluorescence microscope.In control group, the average neuron neurite length was 97.80±6.97(μm/cell) three days after the neural stem cell began to differentiate. The differentiated neuron percentage (NP) was 34.73±5.21%; the percentage of neurite possessing neuron (NPNP) was 58.67+4.31%. In Nogo-P4 group, the average neurite length was 80.54±6.75(μm/cell). NP was 38.97±5.79%, NPNP was 88.52±3.96%. The neurite length has significant difference between the Nogo-P4 group and control group, P＜0.01. The neurite length was shortened about 17% in Nogo-P4 group. So Nogo-P4 inhibits neurites outgrowth during the differentiation progress of neural stem cell. NP has no differences between control group and Nogo-P4 group, while NPNP of Nogo-P4 group is significantly higher than that of control group (p＜0.05).3. siRNA-mediated knockdown of NgR on NSCsAfter transfection, most NSCs survived. Some NSCs were transfected with fluorescence labeled dsRNA (Block-It Fluorescent Oligo) at same condition to observe the transfection efficiency. The total transfection efficiency was 94.3%.We tested the NgR immunoreactivity at 1, 3 and 5 days after transfection. NgR immunoreactivity was markedly reduced in NSCs transfected with siRNA sequence C. The scrambled NgR siRNA sequence did not alter NgR immunoreactivity.In Western blot test, we detect the expression of NgR 1d after transfection of sequence A～C and scrambled siRNA. NSCs without transfection were detected too as control group. NgR gene was knocked down by the three siRNA in different degree. NgR protein was expressed significantly in control group and NSCs transfected by scrambled siRNA.The knockdown efficiency decreased as time elongated after transfection. The most effective siRNA (Sequence C) almost completely knocked down NgR (90.35±3.1% knockdown, P＜0.01vs scrambled siRNA) 1d after transfection and kept high knock-down efficiency (85.22±3.1%) even at 5 days after transfection.The NgR^- NSCs were defined as siRNA group and differentiated in the presence of 10% FBS. After 3 days, the neurite length was 92.14±7.27 (μm/cell); the differentiated neuron percentage (NP) was 32.01±4.82%; the percentage of neurite possessing neuron (NPNP) was 53.62±6.53 %.4. Disinhibition of neurite outgrowth of neurons differentiated from neural stem cells by siRNA-mediated knockdown of NgRIn siRNA+Nogo-P4 group NSCs were transfected with siRNA sequence C to knockdown NgR before they differentiate in the presence of Nogo-P4 and 10% FBS. The neurite length was 94.01±8.37 (μm/cell) three days after the neural stem cell began to differentiate. It has no difference with control group and has significant difference with Nogo-P4 group, P＜0.01. NP was 22.74±4.56%; NPNP was 86.61+2.94 %. Significantly, NPNP of siRNA group was higher than NPNP of control group (p＜0.05) and has no difference with that of Nogo-P4 group.Conelusion1. Neural stem cells express NgR apparently.2. Neural stem cells express NgR apparently during the differentiation progress to neurons.3. Nogo-P4 could inhibit neuron neurite outgrowth differentiated from neural stem cell by interacting with NgR.4. The inhibition effect of Nogo-P4 reached apex in 4μmol/L.5. NgR expression of neural stem cells could be knocked down effectively by RNAi.6. Inhibition effect of Nogo-P4 could be removed by siRNA mediated knockdown of NgR.