| ObjectiveThe mechanism of Nogo-66 inhibition of axon regeneration remains poorly understood, studies of microtubule-affinity regulating kinase 2 (MARK2) in neurite outgrowth have rarely been reported, and the mechanism involved in the Nogo-66 inhibition signaling pathways has not been investigated. This study aimed to determine the role of MARK2 in Nogo-66 inhibition and the effects of MARK2 on axonal regeneration in primary cultured neurons, as well as rats with spinal cord injury, and to provide a theory for approaching the optimum access blocking myelin inhibitors.Methods1. DNA sequences of MARK2were selected from Genbank to design candidate small hairpin (sh) RNAs (shRNA MARK2) and to synthesize the over-expression MARK2 plasmids. The shRNA MARK2 plasmids and MARK2 plasmids were packaged into recombinant adeno-associated virus (AAV) to create shRNA MARK2-AAV and MARK2-AAV. These constructs were sequenced.2. The N2a cells were pass-aged, Nogo-66 (15ng/ml) was added to the medium and maintained for 15min?30min?6h?24h?48h. The protein levels of MARK2 and p-Ser212 were detected via western blot3. The effects of shRNA and MARK2-AAV on the expression of MARK2 were detected by western-blot and immunofluorescence assay in N2a cells.4. The relationships between MARK2 and microtubule associated protein tau, p-S262 tau and MAP1-b were detected by western-blot via MARK2-AAV and shRNA MARK2-AAV5. In vitro, primary cultured rat cortical neurons were divided into for groups:HK+DMSO, OE+DMSO, HK+Nogo-66, OE+Nogo-66; according to the medium with DMSO or Nogo-66, and different kinds of vectors:EGFP-AAV (HK), MARK2-AAV (OE). The axon lengths of e neurons were examined by immunofluorescence microscope and were then stastically analyzed.6. N2a cells were infected by MARK2-AAV, tyrosinated and acetylated ?-tubulin protein levels were detected by western-blot and immunofluorescence.7. Twenty-four adult female Sprague Dawley rats with complete T8 spinal cord transection to establish a rat model of spinal cord injury; and were divided into control, SCI Id, SCI 3d, SCI 7d, SCI 14d, SCI 21d groups (n=4), the leision area of the spinal cord (or normal control group T8 spinal cord) was acquired, the expression changes of MARK2 and p-Ser212 was detected by western-blot.8. Sixteen adult female SD rats, four rats as normal control, twelve rats with SCI were randomly divided into three groups:Saline, EGFP-AAV, MARK2-AAV (4 rats per group),7 days after SCI, four sites of the cortex sensorimotor were injected into saline, EGFP-AAV, MARK2-AAV; and after 14 days the same position were injected with BDA (anterograde labeling),21 days after SCI, all the rats were perfused with PFA, the brains and spinal cord were acquired in normal control, spinal leision was acquired in SCI group, BDA conducting and axon regeneration in the spinal leision were detected by immunofluorescence microscopy.9. The data are represented as means ± standard errors of the means (SEMs). The experiments that involved a single comparison between two groups were analyzed with Student's t-test, whereas differences between multiple groups were analyzed with one-way or two-way analysis of variance (ANOVA). Because of different analysis data, Dunn's posttest, Tukey's posttest and Bonferroni posttest analysis were adopted respectively. P<0.05 was considered statistical significantResults1. The recombinant adenovirus shRNA MARK2-AAV and MARK2-AAV were identified and sequenced by enzyme digestion and sequencing. The results were in complete agreement with the design sequence, the gene sequence was accurate and the recombinant adenovirus was constructed successfully.2. After the addition of Nogo-66, the amount of total MARK2 and the degree of phosphorylated MARK2 (p-Ser212) were changed, the level of MARK2 and p-Ser212 were significantly decreased, at 24h and 48h point, the difference were statistically significant (P<0.05).3. By western-blot, the OE group expressed significantly increased levels of MARK2 (P<0.05) compared with the control group, the shRNA group exhibited significantly lower MARK2 level compared with the control group (P<0.01). Immunocytochemistry experiments (semi-quantitation) indicated that the fluorescence of MARK2 was substantially brighter in the OE group and darker in the shRNA group compared with the control group4. MARK2 reflected the expression of MAP1-b and tau and also its activity (p-S262 tau). MARK2-AAV significantly increased the levels of MAP1-b, tau, and p-S262 tau (P<0.05, comparison between OE and control groups), whereas shRNA MARK2-AAV significantly reduced the levels of MAP1-b, tau, and p-S262 tau (P<0.05, comparison between shRNA and control groups); which coincided with MARK2 expression.5. The axon outgrowth in normal conditions (HK+DMSO) was 68.4±12.3 ?m; in the OE+DMSO group, the axon length was 117.4±22.6 ?m (P<0.05, compared to HK+DMSO group), which indicated the promoting effect of MARK2 on axon length. Whereas, treatment with Nogo-66 alone (HK+Nogo-66) significantly inhibited axon outgrowth down to 28.4±6.3 ?m(P<0.05, compared to HK+DMSO group), which indicated the neurite inhibition effect of Nogo-66. Moreover, pretreatment with MARK2-AAV (OE+Nogo-66) significantly ameliorated the inhibition of neurite outgrowth induced by Nogo-66 to 66.8±9.4 ?m (P<0.05, compared to the HK+Nogo-66 group).6. The Tyr level in the OE group exhibited a significantly increasement compared with the control group (P<0.05). However, the amount of Ace in the OE group was no significantly different compared with the control group.7. Both MARK2 and phosphorylated degree of MARK2 (p-Ser212) exhibited significantly decreased expression in the spinal cord lesion after SCI compared with the No SCI groups (P<0.05).8. Sagittal slices of brain and spinal cord showed that adenovirus and BDA were absorbed and conducted by neurons; BDA anterograde tracing showed that, MARK2-AAV significantly increased the axon length (the percentages of labeled axons) compared with the EGFP-AAV and Saline groups (P<0.05). The axon length was elongated by as much as 600 ?m in the MARK2-AAV group, whereas the maximum length in the EGFP-AAV or saline group was 450 ?m. In addition, the ratios of the axons labeled in the MARK2-AAV group at the 200 ?m,300 ?m,400 ?m point, were significantly different compared with the EGFP-AAV and saline groups (P< 0.01).ConclusionOverall, these novel findings demonstrated that Nogo-66 significantly decreased the expression of MARK2 and the degree of phosphorylated MARK2 at (Ser212), MARK2 was involved in Nogo-66 inhibition by regulating tau and MAP1-b. Overexpression of MARK2 promted the axon outgrowth by increasing the level of tyrosinated a-tubulin, and could significantly amolidate the inhibition effect of Nogo-66. SCI reduced the amount of MARK2 and phosphorylated degree of MARK2 (p-Ser212) in the spinal cord lesion, and MARK2 overexpression promoted axonal regeneration in rats with SCI. Thus, the intracellular MARK2/MAPs/a-tubulin pathway appears to be essential for axonal growth regulation by the Nogo-NgR axis. A regeneration strategy accomplished via MARK2 overexpression may be applicable to promote successful regeneration following many types of central nervous system injuries. |
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