Effects of Alpha-Synuclein on Primary Spinal Cord Neurons Associated with Apoptosis and CNTF Expression

Abstract

Spinal cord injury (SCI) often causes neurological deficits with poor recovery; the treatment, however, is far from satisfaction, and the mechanisms remain unclear. Using immunohistochemistry and western blotting analysis, we found α-synuclein (SNCA) was significantly up-regulated in the spinal caudal segment of rats subjected to spinal cord transection at 3 days post-operation. Moreover, the role of SNCA on neuronal growth and apoptosis in vitro was determined by using overexpressing and interfering SNCA recombined plasmid vectors, and the underlying mechanism was detected by QRT-PCR and western blotting. Spinal neurons transfected with SNCA-shRNA lentivirus gave rise to an optimal neuronal survival, while it results in cell apoptosis in SNCA-ORF group. In molecular level, SNCA silence induced the up-regulation of CNTF and down-regulation of Caspase7/9. Together, endogenous SNCA plays a crucial role in spinal neuronal survival, in which the underlying mechanism may be linked to the regulation both apoptotic genes (Caspase7/9) and CNTF. The present findings therefore provide novel insights into the role of SNCA in spinal cord and associated mechanism, which may provide novel cue for the treatment of SCI in future clinic trials.

Keywords: Alpha-synuclein; Apoptotic gene; Ciliary neurotrophic factor; Primary spinal neurons; Spinal cord transection.

Fig. 1

The expression of SNCA increased in spinal neurons in SCT rats. a Representative photomicrographs of neurons double labeled by NeuN and SNCA in the caudal end of spinal cord. Green signal represented NeuN-positive neurons, red labeled SNCA-positive cells and DAPI (blue) represented nucleus of all cell types. The white scale bar is 250 μm, and the blue scale bar is 50 μm. b The fluorescence intensity of SNCA in the whole spinal gray matter was evaluated by Leica DMI6000B (LAS AF system) and quantified. Data are represented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001. c Western blotting confirmed the expression of SNCA was significantly increased in the caudal end of spinal cord of SCT rats at 3 dpo compared with sham group. Data are represented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Color figure online)

Fig. 2

NeuN and TUNEL double-labeled cells in the caudal end of spinal cord. a Representative photomicrographs of neurons double labeled by NeuN and TUNEL in the caudal end of spinal cord including anterior and posterior horn. Green signal represented NeuN-positive neuron, TUNEL (red) labeled apoptotic cells and DAPI (blue) represented nucleus of all cell types. Scale bar is 25 μm. b The number of NeuN and TUNEL double-positive cells in the anterior horn was evaluated by Leica DMI6000B (LAS AF system) and quantified. Data are represented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001. c The number of NeuN and TUNEL double-positive cells in the posterior horn was evaluated by Leica DMI6000B (LAS AF system) and quantified. Data are represented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 (Color figure online)

Fig. 3

Effects of SNCA-ORF/shRNA lentivirus on the morphology of spinal neurons. a Effects of SNCA-ORF/shRNA lentivirus transfection on the morphology of spinal neurons were shown in 2-day group and 4-day group. Scale bar is 50 μm. b The number of neurons was decreased 2 and 4 days after SNCA-ORF lentivirus treatment but increased following SNCA-shRNA lentivirus transfection measured by Leica DMI6000B (LAS AF system) and quantified after transfected 2 days and c 4 days. The beneficial effect of down-regulated SNCA, inhibition neuron death, was more significant when compared with up-regulation of SNCA (P < 0.01). Six groups respectively were Normal group, PB group (polybrene treated group), NC-ORF group (neurons infected with lentivirus carrying empty vector of SNCA-ORF which expresses GFP but no SNCA), NC-shRNA group (neurons infected with lentivirus carrying shRNA vector containing scramble sequence which were also expressed mcherry), SNCA-shRNA group and SNCA-ORF group. Arrows point to the spinal cord neurons. Data shown are the results of three independent experiments and represented as mean ± SEM. Scale bar represents 50 μm. *P < 0.05; **P < 0.01

Fig. 4

mRNA expression of CNTF and apoptotic signals were regulated by SNCA in spinal neurons. a 4 days after spinal neurons were transfected with lentivirus, the expression of SNCA was dramatically decreased in SNCA-shRNA group, and remarkably increased in SNCA-ORF group. b The expression of neurotrophic factor CNTF was significantly increased in SNCA-shRNA group. c, d respectively showed that the expression of apoptotic genes Caspase7/9 was significantly increased in SNCA-ORF group. Data represent the average from three independent experiments, and were reported as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001

Fig. 5

Protein expression of CNTF and apoptotic factors were regulated by SNCA in spinal neurons. a SNCA, CNTF and Caspase7/9 protein expression in spinal neurons were exhibited. β-actin, a house-keeping gene, was used as internal standard. Lane 1–6 displayed the protein bands of normal group, PB group, NC-ORF group, SNCA-ORF group, NC-shRNA group and SNCA-shRNA group, respectively. Quantitative analysis of SNCA, CNTF, Caspase7 and Caspase9 by western blot was respectively showed in b, c, d and e. The expression of CNTF was significantly increased, contrast to significantly decreased apoptotic genes Caspase7/9 in SNCA-shRNA group. Data represent the average from three independent experiments, and were reported as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001